Whey Protein Films and Coating-A Review

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Whey protein

162 amino acids, 9 -strands, 1 -helix, 2 disulfides, 1 free sulfhydral group (-SH) -barrel domain hydrophobic pocket
Whey Protein characters
Littleher curds and whey
Along came a spider and sat down beside her
and frightened Miss Muffet away
The end!
Thanks for everybody!
It is a rich source of the essential amino acids needed on a daily basis by the body.
Whey protein has the highest value in providing branched-chain amino acids, which result in building and retaining muscle tissue.
Whey protein
What and where is whey protein?
Whey protein is a pure, natural, high quality protein. It comes from milk. During the process of turning milk into cheese, whey protein is separated out.
Whey protein classically a ‘waste product’ of cheese manufacture

人参皂苷治疗骨性关节炎的研究进展

特产研究163Special Wild Economic Animal and Plant ResearchDOI：10.16720/ki.tcyj.2023.093人参皂苷治疗骨性关节炎的研究进展郭校妍1，张伟东1，张扬1※（吉林大学药学院，吉林长春130021）摘要：人参在防治关节软骨损伤退变及参与体外培养软骨细胞修复关节软骨缺损中具有较好治疗前景。

人参皂苷作为人参的主要药理活性成分，在治疗骨性关节炎的进程中发挥关键作用。

人参皂苷根据不同的结构被分为不同的类型，各类型均含有多种人参皂苷单体成分，其治疗骨性关节炎的机制也各不相同。

本文对不同人参皂苷单体治疗骨性关节炎的研究进行梳理和总结，探讨其治疗骨性关节炎的潜在可能性和作用机制，为后期临床应用提供依据。

关键词：骨性关节炎；人参皂苷；信号通路中图分类号：R285文献标识码：A文章编号：1001-4721（2023）03-0163-06Research Progress of Ginsenosides in the Treatment of OsteoarthritisGUO Xiaoyan1,ZHANG Weidong1,ZHANG Yang1※（School of Pharmaceutical Sciences,Jilin University,Changchun130021,China）Abstract:Ginseng has pharmacological effects such as anti-inflammatory,antioxidant,antidepressant,anti-Alzheimer's and anti-athero-sclerosis.Current studies have found that it has good therapeutic prospects in preventing degeneration of articular cartilage damage and parti-cipating in in vitro culture of chondrocytes to repair articular cartilage defects.Ginsenosides,as the main pharmacological active component of ginseng,also play an important role in the process of treating osteoarthritis.Ginsenosides can be classified into different types because of their different structures,and each type contains a variety of ginsenoside monomer components with different mechanisms for the treatment of osteoarthritis.In this paper,we review the research progress of different ginsenoside monomers in the treatment of osteoarthritis,and ex-plore their potential possibilities and mechanisms for the treatment of osteoarthritis,so as to provide a basis for later clinical application. Key words:osteoarthritis;ginsenosides;signaling pathway骨性关节炎（Osteoarthritis,OA）是一种退行性病变，系由于增龄、肥胖、遗传、劳损、创伤、关节先天性异常和关节畸形等诸多因素引起的关节软骨退化损伤、关节边缘和软骨下骨反应性增生。

Protein A, Protein G, and Protein 原理及应用

Chapter24Analysis and Puriﬁcation of Antibody Fragments Using Protein A,Protein G,and Protein LRemko Griep and John McDougall24.1IntroductionToday,monoclonal antibodies(mAbs)form the largest category of biopharmaceu-ticals in clinical trials,and their number is expanding rapidly(DataMonitor2007a,b). The antibodies or functional antibody fragments are being produced not only in artiﬁcial production systems such as mammalian cells,yeast,bacteria,and plant cells but also in transgenic animals such as goats,sheep,and cows.Regardless of the production method,the quality control demand is the same for all of them.Host cell proteins,cell culture media additives,DNA,and endotoxins have to be removed from the mAb preparation to allow the proteins to be safely applied for human therapy.Moreover,antibody aggregates,clipped and low molecular weight species, should also be removed.Several proteins with an inherent afﬁnity for immunoglobulins(Ig)have been isolated from various bacteria.These molecules include protein-A,derived from Staphylococcus aureus(Forsgren and Sjo¨quist1966);protein-G,derived from a group-C Streptococcus(Bjo¨rk and Kronvall1984);andﬁnally protein-L,derived from Peptostreptococcus magnus(A˚kerstro¨m and Bjo¨rk1989;Housden et al.2003, 2004).They all contain repetitive55–76amino acid residues(Fig.24.1)that mediate the actual Ig binding(Kastern et al.1992).The recombinant protein-L can be produced at a yield of up to3g/L in pilot-scale studies.It yields a highly pure,stable,and active protein-L fraction after puriﬁcation,which is binding efﬁciently to most of the human antibodies of the Kappa isotype(Fig.24.2).Protein-G binds not only to the Fc-region but also to the CH1-domain of the human IgG1-isotype.Therefore,it has a broader application compared to protein-A. Some academic groups have also reported the use of genetically fused protein-LG (Kihlberg et al.1996;Harrison et al.2008)or protein-AG(Eliasson et al.1988; R.Griep(*)and J.McDougallAfﬁtech AS,Gaustadalle´en21,Oslo3490,Norwaye-mail:r.griep@afﬁ301 R.Kontermann and S.Du¨bel(eds.),Antibody Engineering Vol.2,DOI10.1007/978-3-642-01147-4_24,#Springer-Verlag Berlin Heidelberg2010Bergmann-Leitner et al.2008)and protein-LA (Svensson et al.1998)for monoclo-nal antibody puriﬁcation.They indeed obtained broader functional ligands because the binding characteristics of both parental proteins were maintained.The ability of protein-A,-G,or -L to maintain their functionality,on conjugation with ﬂuoro-chromes,enzymes (Fig.24.3a ),or gold particles,makes them highly valuable secondary reagents for the detection of primary antibodies in ELISA,immunohis-tochemistry,ﬂowcytometry,and electronmicroscopy.Protein-A mainly binds to the Fc-region of the IgG from several human isotypes (Table 24.1)but only to a single variable region of the heavy-chain family (Starovasnik et al.1999).In contrast,protein-L binds to most of the human Kappa light-chains of the k I,k III,and k IV families.These comprise 55–60%of all IgA,IgE,and IgM antibodies in the human serum (Solomon 1976)and can thus be used to purify all monoclonal antibodies of those Kappa sub-types (Nilson et al.1992)or fragments derived thereof.This,without the need to genetically engineer afﬁnity-tags onto the protein of interest (Devaux et al.2001;Das et al.2005;Cossins et al.2007).The k antibodies described in Fig 24.3b were originally derived from a large human unbiased antibody phage library (Løset et al.2005)and six out of the ten k antibodies strongly react with protein-L (Fig.24.3b ).These authors also demon-strated that preselection of this particular phage-library for the binding to protein-L can be of use.It yields phage-antibodies with improved functionality,as each phage is actually assayed for its ability to express at least one functional scFv on its surface prior to its selection against an antigen.An alternative approach is to build a highly diverse library,on the basis of certain well-expressing and protein-L binding Kappa light-chain genes (Holt et al.2008).Moreover,protein-L has a clear advantage over protein-A and protein-G,as it does not bind to bovine IgG or to bovine serum albumin.This might be of major importance when one is forced to use bovine serum as additive to the cell culture medium to prevent certain types of mammalian cells from dying.Thus far,protein-L has not been available for theindustrial-scale117824473169575239Fig.24.1Structure of the protein-L molecule comprising 719amino acids.The numbers,indicat-ing the amino acids of the beginning of each domain,are listed below the boxes.Included are the signal peptide (SP),the signal peptide cleavage site is indicated by the arrow,the NH 2-terminal(A),the repeated units with Ig-binding activity (B1–B5),the spacer region (S),the repeats (C),the wall spanning domain (W),and the transmembrane region (M).The recombinant protein-L consists of four Ig-binding domains (B1–B4),which can bind to the Kappa region without interfering with the antigen-binding site of the immunoglobulin302R.Griep and J.McDougallpuriﬁcation,but recently,a development toward introduction into the bulk market has been initiated.A prerequisite for (cost)-efﬁcient industrial-scale puriﬁcation of MAbs is that the ligands like protein-A,protein-L,and protein-G can be coupled efﬁciently to solid matrices like controlled pore glass (Millipore)and to agarose with varying degrees of cross-linking (GE Healthcare).These materials are rigid and can be operated at high ﬂow velocities.Highly porous materials exert a low-pressure drop,a low mass transfer resistance,and a high dynamic capacity (LeVan et al.1997).Unfortunately,these features are nonexclusive to a certain extent.A highly porous medium could have a low equilibrium capacity because of a limited surface area and simulta-neously have good mass transfer characteristics but bad ﬂow properties as a result bm g /m L Protein-L 2341-+ -+ IgG 101520253750kDa751342ac Tim e after induction Fig.24.2(a )A pilot-scale production system has been set up for production of recombinant protein-L in E.coli .The DNA sequence encoding the B1-4domains has been cloned into a pJB-vector (Sletta et al.2004)and the recombinant protein-L was expressed intracellular in high-cell-density-cultivation as shown here for ﬁve separate cases.The produced protein-L was extracted from the cytoplasm,puriﬁed,and analyzed by SDS-PAGE.(b )SDS-PAGE analysis of the puriﬁed protein-L (c )CNBR activated-sepharose beads were conjugated without (À)and with polyclonal human IgG (þ)and incubated with protein-L preparations which were stored for 1month,either at 4 C (lane 1and 2)or at 37 C (lane 3and 4).Subsequently,the obtained supernatants were analyzed by SDS-PAGE for the presence of unbound protein-L.As can be observed from this picture,the majority of the protein-L is speciﬁcally binding to the polyclonal IgG,even after storage for 1month at 37 C24Analysis and Puriﬁcation of Antibody Fragments 303of its softness.In contrast,a resin with a high equilibrium capacity might have increased mass transfer resistance.As the costs of resins are high,the ligands should maintain their selectivity and have good chemical stabilities over a long period of time.Cleaning in place procedures (CIP)with repeated alkaline exposures can be detrimental for ligands like protein-A and protein-G.To facilitate CIP,some of the ligands,such as MabSelect (GE Healthcare)or a protein-A analog Z(F30A)(Linhult et al.2004),could be optimized and are now available as an improved alkaline resistant alter-native for protein-A.Also,for protein-G,an improved mutant was engineered (Gu¨lich et al.2002),while according to the results of Enever (Enever et al.2005),higher afﬁnity variants can also be expected for protein-L.Because of the acidic elution and the high concentration of Mabs on the column,aggregates are easily formed (Shukla et al.2007).In addition,leaching and cleavage of the ligand is observed for protein-A (Carter-Franklin et al.2007)and protein-G.As a consequence,both aggregates and leached ligand have to be removed from the a0.40.81.21.6O D 405Applied antibody0.51.01.52.02.53.0I g G -1I g G -2I g G -3I g G -4I g G -5I g G -6I g G -7I g G -8F ab -1sc F v -1c o n t r o l O D 405bFig.24.3(a )Quality control of the produced recombinant protein-L with the aid of an ELISA.A maxisorb ELISA-plate,coated with human IgG,was preincubated with different concentrations of unconjugated recombinant protein-L (rProtein-L TM ,#101Actigen)prior to incubation with a protein-L/HRP conjugate (rProtein-L TM HRP,#301Actigen).After washing,chromogenic sub-strate was added and the absorbance of the individual wells was measured at OD 405nm.The signal shows clear inhibition by the unconjugated protein-L (b )A maxisorb ELISA-plate,coated with different human IgG(k )antibodies,a human Fab(k )or with a scFv(k )fragment (all at 0.1m g/well)was incubated with a protein-L/HRP conjugate.After washing,chromogenic substrate was added and the absorbance of the individual wells was measured at OD 405nm304R.Griep and J.McDougallTable24.1Binding of immunoglobulin isotypes and some of their smaller derivatives to protein-A,protein-G,protein-L,protein-AG protein-LG,and protein-LA,on the basis of data that were obtained from Pierce;GE healthcare;Bonifacino,and Dell’Angelica1998;Hober et al.2007; Kihlberg et al.1996;de Chaˆteau et al.1993,and Svensson et al.1998.(?:unknown,À¼no binding,Æ¼very low binding,þ¼low binding,þþ¼good binding,þþþ¼high binding, V H3and K k=binding only to these speciﬁc human heavy-and light-chain families)Species Subclass Prot-A Prot-GProt-L Prot-AGProt-LGProt-LAHuman IgG1þþþþþþþþ(k)þþþþþþþIgG2þþþþþþþþ(k)þþþþþþþIgG3–þþþþþ(k)þþþþþþIgG4þþþþþþþþ(k)þþþþþþþIgE V H3–þþ(k)V H3þþþ(k)þþþ(k)(V H3)IgA V H3–þþ(k)V H3þþþ(k)þþþ(k)(V H3)IgM V H3–þþ(k)V H3þþþ(k)þþþ(k)(V H3)Human Antibody fragments Lamdda-LC––––––Kappa-LC V H3–þþ(k)V H3þþþ(k)þþþ(k)(V H3) IgG1-Fab V H3þþþþþ(k)V H3þþþ(k)þþþ(k)(V H3) Fv V H3–þþ(k)V H3þþþ(k)þþþ(k)(V H3) scFv V H3–þþ(k)V H3þþþ(k)þþþ(k)(V H3) single domain V H3–þþ(k-LC)V H3þþþ(k)þþþ(k)(V H3)Mouse IgG1þþ35%of total IgGin mouse sera þþþþþIgG2aþþþþþþþþþIgG2bþþþþþþþþþIgG3þþþþþþþGuinea pig IgG1þþþþ<10%of total IgG?þþþþIgG2þþþþ?þþþþBovine IgGþþþþ–þþþþþþþCat IgGþþþþ?þþþþþþChicken IgYÆþ>50%of total IgGÆþ>50%oftotal IgG Dog IgGþþþþþ–þþþþþþþDonkey IgG–þþ??þþ?Hamster IgGþþþþþþþþþþþþþþHorse IgGþþþþþ–þþþþþþþGoat IgGþþþ??þþþþþMonkey IgGþþþþþþ?þþþ??Pig IgGþþþþþþ50%of total IgGþþþþþþþþRabbit Nodistinctionþþþþþþ–þþþþþþþþRat IgGþþþ35%of total IgGþþþþþþSheep IgGþþþ?þþþþþþ24Analysis and Puriﬁcation of Antibody Fragments305306R.Griep and J.McDougall antibody preparation before it can be applied.Therefore,IgG puriﬁcation with protein-A,-L,or-G is usually only theﬁrst step and is usually followed by a series of multiple polishing steps.A combination of anion exchange chromatography in ﬂow through mode and cation exchange chromatography removes host cell proteins, DNA,endotoxins,leached protein,and aggregates efﬁciently(Tugcu et al.2007).Despite the wide variety within the applied monoclonal antibodies,such as, chimeric,humanized,and fully human IgGs of various isotypes,a general puriﬁca-tion strategy is desirable.To date,several comparative studies are available in the literature(Fuglistaller1989;Fahrner et al.1999;Godfrey et al.1993;Hahn et al. 2003,2006;Ghose et al.2007;Swinnen et al.2007and Katoh et al.2007),but new matrices are available to be introduced on theﬂourishing antibody market(Boi et al.2008).In addition,a total matrix free puriﬁcation method has been described (Kim et al.2005),which is on the basis of a reversible temperature triggered precipitation of antibodies with the aid of protein-L,or protein-LG fused to elastin-like proteins.The basic protocols for protein-A,protein-L,and protein-G chromatography are relatively straightforward.Bind the immonoglobulins at a neutal pH and elute at an acidic pH.Salt ions even promote binding of IgG to protein-A.Often a stationary phase is employed for the puriﬁcation of multiple monoclonal antibodies and although the Fc region is the same,still different binding and elution parameters might have to be established for different variable regions(Ghose et al.2005, 2007).As demonstration,methods are described for the puriﬁcation of polyclonal human IgG/k from serum IgG,a scFv(k)and a IgG1derived CH1/l Fab fragment from an E.coli extract using protein-L and protein-G,respectively.Despite the described differences in the literature between unique human IgG molecules,the puriﬁcation methodology described below will yield pure,homogeneous,and highly active antibody preparations for almost any antibody without any major changes to these protocols.24.2Puriﬁcation of Human IgG/k Antibody Fragmentswith Protein-LFor the isolation of a polyclonal IgG fraction from a human serum or of an scFv fragment from bacterial periplasmic preparation,protein-L is known to be an excellent ligand(Fig.24.4).The isolated IgG and scFv have a high purity and the puriﬁcation method,as described below,is easy to use.24.2.1Materials–Protein-L agarose slurry(rProtein-L TM–agarose,#201,Actigen)in50%ethanol;maximum binding capacity is10mg IgG per mL beads–Human serumabcFig.24.4Representative examples of the versatile application of protein-L.(a)Puriﬁcation of Polyclonal antibodies from human serum.The pooled fractions are indicated with the double arrow;the solid lines indicate the optical density at 280nm,whereas the dotted lines reﬂect the pH.(b)Separation of a protein-A puriﬁed human IgG preparation in a Kappa and Lambda fraction via protein-L.(c)Puriﬁcation of a Kappa scFv from a bacterial extract on a protein-A column24Analysis and Puriﬁcation of Antibody Fragments 307308R.Griep and J.McDougall –PBS–Elution buffer(0,1M Glycin-HCl,pH2,5)–Neutralizing buffer(1M Tris-HCL,pH9,0)–Polystyrene columns,2mL(Pierce,#29920)–20%Ethanol–Deionised water24.2.2Method1.Set up a2mL column and load with0.5mL Protein L-agarose(thus1mL as in50%volume with ethanol/PBS).2.Wait until the gel is settled and wash with5mL PBS.3.Load5mL IgG-solution.4.Collect the IgGﬂow through fraction.5.Wash with10mL PBS.6.Collect the wash fraction.7.Add350m L1M Tris-HCl,pH9.0to the tubes in the fraction collector prior toelution to immediately neutralize the sample upon elution.8.Elute with5mL elution buffer.9.Collect the eluate.10.Wash the column with5column volumes of deionised water.11.Wash the column with5column volumes of20%ethanol,and store it at4 C.12.Dilute the eluate,ﬂow through,wash,and eluted fraction1:10with PBS.13.Determine the absorbance at280nm.14.Analyze the purity of the sample by SDS-PAGE.24.3Puriﬁcation of a Monoclonal Human IgG Fab Fragmentwith Protein-GThe isolation of recombinant Fab fragments from bacterial extracts requires a more demanding puriﬁcation procedure because the heavy-and light-chain frag-ments are not produced in equal amounts.In general,the light-chain is produced at higher levels and secreted as a contaminating light-chain dimer.Therefore,the isolation procedure has to consist of two subsequent steps.Theﬁrst step is isolation of all the light-chains via a his-tag,which is located on the C-terminus. This is followed by an afﬁnity puriﬁcation of the heavy-chain fragment via protein-G,which binds to the CH1-region of human IgG1.As a consequence,24Analysis and Puriﬁcation of Antibody Fragments309 all light-chain dimers will be removed during the procedure described below, which is easy to use and will yield high quality Fab fragments(Fig.24.5).24.3.1Step1:Ni-IMAC Puriﬁcation of a Fab Fragment24.3.1.1Materials–A¨kta TM Puriﬁer–A¨kta column HisTrap TM FF,1mL(GE Healthcare)–20%Ethanol–Deionised water–0.8m m,0.45m m and0,20m mﬁlters–2M Imidazole,pH7.0(Preferably from Fluka,sold by Sigma-Aldrich,ultra-pure,cat.no56749,which has no interfering absorbance at280nm)–Buffer-A:IMAC loading buffer(20mM sodium phosphate,500mM NaCl,pH7.4)–Buffer-B:IMAC Elution buffer(20mM sodium phosphate,150mM NaCl, 500mM Imidazole,10%glycerol,pH7.4)–Dialyzed periplasmic E.coli extracts24.3.1.2Method1.Filter all the buffers through a0.20m mﬁlter.2.Preferably precool the buffers at4 C.3.Filter the pooled and dialyzed periplasmic fractions through0.8and0.45m mﬁlters before loading it onto the IMAC column.4.Add500m L of the2M imidazole stock per100mL of theﬁltered periplasmicfraction to obtain aﬁnal concentration of10mM.5.Equilibrate the column with5column volumes of buffer-A.6.Load the sample on the column.7.Wash the column with20mM imidazole until the unbound proteins have beenwashed out of the column(5column volumes)and the OD280signal has returned to the baseline.8.Elute with100%Buffer-B.9.Wash the column with5column volumes of deionised water.10.Wash the column with5column volumes of20%ethanol,and store it at4 C.11.Optional:analyze the isolated fractions by SDS-PAGE before pooling.12.Avoid freezing samples with imidazole,as it has been observed that this canseverely decrease the activity of the puriﬁed antibody fragments.310R.Griep and J.McDougall abcFig.24.5Representative example of the protein-G puriﬁcation of a monoclonal human IgG1/l Fab fragment from the eluent of a nickel-NTA column.(a)The Fab fragments were isolated from a bacterial extract through the interaction of the His-tag of the light-chain with nickel-NTA beads.24.3.2Step2:Protein-G Puriﬁcation of a Fab Fragment24.3.2.1Materials–A¨kta TM Puriﬁer–Nickel-NTA prepuriﬁed Fab fragments–HiTrap TM_ProteinG_HP_1mL FF,(GE HEALTHCARE)–20%Ethanol–Deionised water–1M Tris-HCl,pH9,0–Loading buffer:20mM sodium phosphate with500mM NaCl,pH7.4–Elution buffer:0,1M Glycine-HCl,pH2.524.3.2.2Method1.Pool the fractions,preferably obtained from a Fab preparation,which wereprepuriﬁed on a nickel-NTA column.2.Wash the general system of the A¨kta Puriﬁer TM as well as the10mL sampleloop with Loading buffer.3.Add300m L Tris-HCl,pH9.0to the tubes in the fraction collector prior toelution to immediately neutralize the samples upon elution.4.Load the dialyzed sample onto the column.5.Wash the column with minimal5column volumes of loading buffer until theunbound proteins have been washed out of the column and the OD280signal has returned to the baseline.6.Elute the captured Fab fragments via elution with100%of the elution buffer.7.Wash the column with5column volumes of deionised water.8.Wash the column with5column volumes of20%ethanol,and store it at4 C.9.To obtain Fab fragments of the highest quality an SDS-PAGE analysis can beperformed before deciding which of the fractions should be pooled.10.Dialyze against PBS containing5%glycerol,preferably at a Fab concentrationbelow1mg/mL;this is to prevent precipitation.<Fig.24.5(continued)The pooled fractions are indicated with the double arrow,and the solid lines indicate the optical density at280nm,whereas the dotted lines reﬂect the pH.(b)The excess of light-chain dimers was removed with the protein-G puriﬁcation step.The pooled fractions are indicated with the double arrow.(c)Analysis by SDS-PAGE under nonreducing conditions,at the left of the marker(M)and under reducing conditions at the right side(lane-1,ﬁrst periplasmic extract1;lane-2second periplasmic extract;lane-3,efﬂuent from the IMAC column;lane4,eluent from IMAC column,also used to load the protein-G column;lane5,efﬂuent from the protein-G column;lane6eluent from protein-G column and lane7,theﬁnal obtained Fab fragment after up concentration and dialyses against PBS).The analysis clearly showed that the light-chain dimer is efﬁciently removed during the protein-G step(lane-5versus lane-6under reducing conditions)and the high purity of the obtained Fab fragment in theﬁnal product11.Determine the protein concentration with a spectrophotometer at OD280.12.Store the samples at4 C(1day)or atÀ20 C for longer periods of time,butstorage atÀ80 C is recommended to guarantee long lasting quality of the puriﬁed Fab fragments.24.4Trouble ShootingIt might be valuable to monitor the binding efﬁciency for each speciﬁc antibody with techniques such as ELISA,SDS-PAGE,and Western blotting.Optimization of the binding properties of,for instance,rProtein L can result in a tenfold higher yield for a particular antibody.Similar optimizations have been reported for protein-G and protein-A with the application of salts such as sodium chloride and sodium sulfate,which favor increases in hydrophobic interactions.In addition, the pH of the loading buffer can be increased from neutral to more basic(pH9)to maximize the yield.In addition,the concentration of the feedstock should be altered for each antibody during the optimization process to gain maximum binding and elution characteristics.In case of problems with serum derived impurities,protein-L performs speciﬁcally in the presence of a large background (up to tenfold)of bovine immunoglobulins.This is particularly valuable when isolating antibodies from culture media containing bovine serum or from the milk of transgenic animals.24.5Concluding RemarksBefore purifying an antibody,regardless the source,consideration should be given to theﬁnal use of the product.For many applications,both monoclonal and polyclonal antibodies may be used in an impure form.However,for conjugation toﬂuorochromes or enzymes,simple ligand-based puriﬁcation is sufﬁcient,but for cell-based assays,a higher level of puriﬁcation is an absolute requirement.In addition,it all depends on the nature of the antibody fragment combined with the method used for its production whether protein A,protein G,protein L or even a combination of these should be used to obtain optimal results.Whichever method is chosen,care should be taken not to expose the antibodies for an extended time to either strong acidic or basic conditions.This can be avoided by adding a neutraliz-ing buffer in the collection tubes prior to the elution step.In addition,buffer conditions with a pH around the isoelectric point might favor precipitation.A general formulation buffer(10mM Na-citrate/pH6containing:300mM sucrose, 0.9%NaCl,50mM glycine,3.5mM methionine,and0.05%polysorbate-80)can be recommended,which prevents precipitation,aggregation,and oxidation of the puriﬁed antibody fragments.Finally,antibody puriﬁcation can be performed withfancy equipment,but this is not at all an absolute requirement to obtain excellent results.Simple gravityﬂow always works,even in the case of power failure. ReferencesA˚kerstro¨m B,Bjo¨rk L(1989)Protein L:an immunoglobulin light chain binding bacterial protein.J Biol Chem264:19740–19746Bergmann-Leitner ES,Mease RM,Duncan EH,Khan F,Waitumbi J,Angov E(2008)Evaluation of immunoglobulin puriﬁcation methods and their impact on quality and yield of antigen-speciﬁc antibodies.Malar J7:129–139Bjo¨rk L,Kronvall G(1984)Puriﬁcation and some properties of Streptococcal protein G a novel IgG-binding reagent.J Immunol133:969–974Boi C,Dimartino S,Sarti GC(2008)Performance of a new protein A afﬁnity membrane for the primary recovery of antibodies.Biotech Prog24:640–647Bonifacino JS,Dell’Angelica EC(1998)Immunoprecipitation.Curr Protoc Cell Biol Chapter 7:7.2.1–7.2.21Carter-Franklin JN,Victa C,McDonald P,Fahrner R(2007)Fragments of protein A eluted during protein A chromatography.J Chromatog A1163:105–111Cossins AJ,Harrison S,Popplewell AG,Gore MG(2007)Recombinant production of a V L single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein L.Protein Expr Purif51:253–259Das D,Allen TM,Suresh MR(2005)Comparative evaluation of two puriﬁcation methods of anti-CD19-c-myc-His6-Cys scFv.Protein Expr Purif39:199–208DataMonitor(2007)Monoclonal Antibodies Report Market Model–Detailed analysis of the monoclonal antibody segment,encompassing market dynamics,key therapy areas,technology and target types through to2012,evaluating the strategies companies are using to capitalize on this lucrative market.Reference Code:IMHC0090,June2007DataMonitor(2007)Monoclonal Antibodies Report Part 1.Reference Code:DMHC2291, June2007De Chaˆteau M,Nilson BH,Erntell M,Myhre E,Magnusson CG,Akerstro¨m B,Bjo¨rck L(1993) On the interaction between protein L and immunoglobulins of various mammalian species.Scand J Immunol37:339–405Devaux C,Moreau E,Goyffon M,Rochat H,Billiald P(2001)Construction and functional evaluation of a single-chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector.Eur J Biochem268:694–702Eliasson M,Olsson A,Palmcrantz E,Wiberg K,Ingana¨s M,Guss B,Lindberg M,Uhle´n M(1988) Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G.J Biol Chem263:4323Enever C,Tomlinson IA,Lund J,Levens M,Holliger P(2005)Engineering high afﬁnity super-antigens by phage display.J Mol Biol347:107–120Fahrner RL,Whitney DH,Vanderlaan M,Blank GS(1999)Performance comparison of protein A afﬁnity-chromatography sorbents for purifying recombinant monoclonal antibodies.Biotech-nol Appl Biochem30:121–128Forsgren A,Sjo¨quist J(1966)Protein A from staphylococcus Aureus I Pseudoimmune reaction with human gamma-globulin.J Immunol97:822–827Fuglistaller P(1989)Comparison of immunoglobulin binding capacities and ligand leakage using eight different protein A afﬁnity chromatography matrices.J Immunol Methods124:171–177 Ghose S,Allen M,Hubbard B,Brooks C,Cramer SM(2005)Antibody variable region interactions with protein A:Implications for the development of generic puriﬁcation process.Biotechnol Bioeng92:665–673。

T7噬菌体DNA的提取及其反向遗传拯救方法的建立

T7噬菌体DNA的提取及其反向遗传拯救方法的建立徐海;王义伟;陈瑾;郑其升;侯继波【摘要】从T7噬菌体培养液中粗提噬菌体颗粒,经热裂解后用苯酚、氯仿抽提进而获得纯净的T7噬菌体DNA.用PCR、酶切法鉴定T7噬菌体DNA的完整性.通过对不同感受态细菌浓度、T7噬菌体DNA用量、电转化电压条件的优化,建立了T7噬菌体反向遗传拯救方法.结果显示,提取的DNA结构完整,能够被特异性酶切割,多克隆位点序列正确.T7噬菌体的反向遗传拯救方法最优化条件为200 ng T7噬菌体DNA、1 ml 5×109感受态细菌、1.5 kV电转化电压,在此条件下获得的拯救效率为3.5×105 PFU/ng (DNA).%The T7 phage DNA was purified with benzene polyphenol and chloroform from T7 phage particles after heat cracking. The integrity of T7 phage DNA was identified by PCR and enzemy digestion, and a reverse genetic rescue system for the purified T7 DNA was established through the optimization of the conditions, such as T7 phage DNA input dosage, density of complete cells, and electrotransformation voltage. The results showed that the purified T7 DNA could be digested with EcoR I or Hind Ⅲ, and the multiclon site was correct. The reverse genetics rescue system was successfully established. The highest rescue efficiency was obtained under the conditions of 200 ng T7 phage DNA,5×l09 complete cell per millili ter and 1.5 kV electrotransformation voltage, with a output of 3.5×l05 PFU/ng (DNA).【期刊名称】《江苏农业学报》【年(卷),期】2012(028)002【总页数】4页(P355-358)【关键词】T7噬菌体;DNA提取;电转化;反向遗传拯救【作者】徐海;王义伟;陈瑾;郑其升;侯继波【作者单位】江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014;江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014;江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014;江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014;江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014【正文语种】中文【中图分类】S432.4+1T7噬菌体是感染大肠杆菌的烈性噬菌体，在大肠杆菌的胞浆内组装，成熟的T7噬菌体通过细胞裂解而释放，现已完成其全序列分析，遗传背景清楚，病毒颗粒结构复杂。

Charge heterogeneity profiling of monoclonal antibodies using low ionic strength IEC

Journal of Chromatography A,1317 (2013) 148–154Contents lists available at ScienceDirectJournal of ChromatographyAj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o maCharge heterogeneity proﬁling of monoclonal antibodies using low ionic strength ion-exchange chromatography and well-controlled pH gradients on monolithic columnsMohammad Talebi a ,Anna Nordborg a ,Andras Gaspar a ,Nathan cher b ,Qian Wang b ,Xiaoping Z.He b ,Paul R.Haddad a ,Emily F.Hilder a ,∗aPﬁzer Analytical Research Centre (PARC)and Australian Centre for Research on Separation Science (ACROSS),School of Chemistry,University of Tasmania,Hobart,Tasmania,Australia bAnalytical R&D,Pﬁzer BioTherapeutics Pharmaceutical Sciences,Chesterﬁeld,MO,USAa r t i c l ei n f oArticle history:Received 13May 2013Received in revised form 13August 2013Accepted 16August 2013Available online 21 August 2013Keywords:pH gradient Ion exchangeMonoclonal antibodies Polymer monolithImaged capillary isoelectric focussingLiquid chromatography–mass spectrometrya b s t r a c tIn this work,the suitability of employing shallow pH gradients generated using single component buffer systems as eluents through cation-exchange (CEX)monolithic columns is demonstrated for the high-resolution separation of monoclonal antibody (mAb)charge variants in three different biopharma-ceuticals.A useful selection of small molecule buffer species is described that can be used within very narrow pH ranges (typically 1pH unit)deﬁned by their buffer capacity for producing controlled and smooth pH proﬁles when used together with porous polymer ing very low ionic strength eluents also enabled direct coupling with electrospray ionisation mass spectrometry.The results obtained by the developed pH gradient approach for the separation of closely related antibody species appear to be consistent with those obtained by imaged capillary isoelectric focusing (iCE)in terms of both resolu-tion and separation proﬁle.Both determinants of resolution,i.e.,peak compression and peak separation contribute to the gains in resolution,evidently through the Donnan potential effect,which is increased by decreasing the eluent concentration,and also through the way electrostatic charges are distributed on the protein surface.Retention mechanisms based on the trends observed in retention of proteins at pH values higher than the electrophoretic p I are also discussed using applicable theories.Employing monolithic ion-exchangers is shown to enable fast method development,short analysis time,and high sample throughput owing to the accelerated mass transport of the monolithic media.The possibility of short analysis time,typically less than 15min,and high sample throughput is extremely useful in the assessment of charge-based changes to the mAb products,such as during manufacturing or storage.© 2013 Elsevier B.V. All rights reserved.1.IntroductionThe advances in biotechnology in the last quarter of the 20th century have led to the development of new technologies for the production of complex biomolecules which could potentially be used in human health care in the areas of diagnostics,prevention and treatment of diseases.Qualities,such as high (target)selec-tivity,the ability to initiate immune recognition of the target,and long circulation half lives,have made the development of human-ised mAbs the fastest growing segment of therapeutic drugs [1,2].In the production of mAbs the ﬁnal product often exhibits a number of variations from the expected or desired structure.These alterations may result from either known or novel types of posttranslational∗Corresponding author.Tel.:+61362267670.E-mail address:emily.hilder@.au (E.F.Hilder).modiﬁcations or from spontaneous,non-enzymatic protein degra-dation which bring about charge and size mon modiﬁcations of the primary sequence include N-glycosylation,methionine oxidation,proteolytic fragmentation,and deamidation [3].It has been shown that charge variants of therapeutic proteins can have signiﬁcantly different bioactivity.For example,Harris et al.[4]showed that deamidated variants of recombinant human mAbs had reduced potency in a bioactivity assay.As protein charge het-erogeneity is an important factor in quality assessment of protein therapeutics,regulatory authorities such as the International Con-ference on Harmonisation (ICH)have set criteria for monitoring and characterising the degree and proﬁle of variations to ensure lot-to-lot consistency and product stability [5].Considering the large size of antibodies and the minor struc-tural diversity between the variants,the existence of these variants imposes a great challenge for their separation.Ion-exchange (IEX)chromatography is a non-denaturing technique used widely to0021-9673/$–see front matter © 2013 Elsevier B.V. All rights reserved./10.1016/j.chroma.2013.08.061M.Talebi et al./J.Chromatogr.A1317 (2013) 148–154149separate and isolate protein charge variants for subsequent charac-terisation.However,when operating under a salt gradient approach (classical mode),IEX chromatography has been shown to exhibit limited selectivity when complex proteins with the same number of effective charges are to be separated[6]and lack of robustness when carboxypeptidase B(CPB)-treated mAbs are to be analysed [7].Capillary isoelectric focusing(CIEF)is another separation tech-nique used frequently to assess charge heterogeneity of proteins in which a complex mixture of ampholytes(polyionic organic elec-trolytes)is used to establish a pH gradient into a capillary with the aid of an electricﬁeld.The electricﬁeld causes protein isoforms to focus along the capillary according to their isoelectric point where they have zero net charge and then mobilise towards an on-column detector located at one end of the capillary.Due to the distortion of the pH gradient,which affects reproducibility in migration time and peak area,the mobilisation step often requires optimisation[8]. The introduction of imaged capillary electrophoresis(iCE),where imaging is performed of an entire capillary,has overcome this issue by eliminating the need for the mobilisation step through single point detection.While CIEF is perhaps the most powerful of the known separation technologies for charge variants,the difﬁculty of collecting fractions when compared to IEX chromatography has conﬁned the method to be suitable for monitoring of variants but not for their preparative separation or isolation(peak identiﬁca-tion)[2,6].Also,some authors believe that while the separations are consistent between the two methods,CIEF is not as precise as IEX chromatography and therefore cannot be considered as a suitable replacement[9].To the contrary,however,some have con-cluded that CE techniques could be superior to IEX chromatography in terms of both separation speed and obtainable high resolution and therefore could constitute a routine tool for assessing charge heterogeneity of proteins[8,10].Developed by Sluyterman et al.[11–15]in the late1970s, chromatofocusing(internal pH gradient)is recognised as the chromatographic analogy to IEF[9],mitigating many of the short-comings of classical IEX chromatography and combining some unique features of both methods.Chromatofocusing has been demonstrated to be useful for separating protein isoforms due to its high resolving power and ability to retain the protein native state[7,16].There are however some limitations to this technique such as the cost of polyampholyte buffers employed,the necessity of column regeneration after each separation,and the inﬂexibility in controlling pH gradient slope[7,17,18].Alternatively,pH gra-dient can be conducted externally by pre-column mixing of two eluting buffers at different pH values consisting of common buffer species.As the slope and proﬁle of the pH gradient can be easily controlled by changing the elution program with less dependence on the buffer composition and column chemistry,this manner of introducing pH gradients should allow for more convenient method development and optimisation[17,18].The externally induced pH gradient has been applied for separation of deamidated variants of a mAb[3],resolving C-terminal lysine isoforms of a mAb after treat-ing with carboxypeptidase B[7]and also for the analysis of charge variants of full-length mAbs[9].Currently,particle-packed columns represent the most common stationary phases for high performance liquid chromatography. Despite immense popularity,their application for rapid and efﬁ-cient separation of macromolecules is not as convenient as for small molecules.This is mostly because of slow diffusional mass transfer of large solutes and also the large void volume existing between the packed particles[19].Additionally,biocompatibility of stationary phases has become a new challenge when analysing biomolecules(including peptide and proteins).As deﬁned by Li et al.[20],a biocompatible stationary phase material should be able to resist non-speciﬁc adsorption of biomolecules and preserve the bioactivity of the target biomolecules.These challenges are well met by employing monolithic media.Mass transfer in monolithic sorbents is mostly dominated by convection,rather than diffusion, and is therefore fast,even for large biomolecules.On the other hand,the expected biocompatibility of the most frequently used polymers in making porous monoliths,i.e.,poly(meth)acrylate and polyacrylamide,make these stationary phases highly suited for use in protein separation applications.We recently reviewed advances in polymer monoliths for IEX chromatography of biomolecules and addressed the importance of reducing non-speciﬁc interactions between analyte and stationary phase[21].While IEX chromatog-raphy of proteins using monolithic columns is frequently seen in the literature[22–24],very little effort has been directed towards employing this technique for separation of large proteins,such as mAbs.In continuing our recent efforts to resolve charge variants of mAbs with the aid of IEX monolithic columns[25];the maximum achievable resolution for mAb isoforms was pursued in this work using CEX columns in combination with simple,yet efﬁcient,buffer systems.Unlike previous reports[6,9,18,26],we operated IEX chro-matography employing shallow pH proﬁles over a limited pH range (typically1pH unit)generated by single component buffer sys-tems at very low ionic strength.The suitability of the proposed buffer system in direct coupling of IEX chromatography to MS was also demonstrated.Due to their size and complexity,mAbs are typically characterised by two or more orthogonal separation methods[9].Therefore,the performance of the developed method was also assessed by comparing the results with those obtained by iCE.It was hoped that similar charge heterogeneity proﬁles could be achieved for mAbs analysed under two different separation mechanisms.2.Experimental2.1.Reagents and chemicalsThe buffering species used in this work,including imidazole,piperazine dihydrochloride hydrate(PDH),and tris(hydroxymethyl)aminomethane(Tris),diethanolamine(DEA) and ammonium hydroxide(AMH),28%(v/v)were all obtained from Sigma–Aldrich(Sydney,Australia)and triethanolamine(TEA) was from BDH(Poole,England).Sodium chloride,hydrochloric acid and sodium hydroxide(98.8%),methanol(LC–MS grade)were also from Sigma.All chemicals were of analytical grade unless speciﬁed otherwise.For iCE experiments,pharmalyte pH3–10,sucrose and urea were obtained from Sigma–Aldrich,while methyl cellulose (1%)and the Chemical Test Kit were from ProteinSimple(formerly Convergent Bioscience,Toronto,ON,Canada).The p I markers including p I s 5.13, 6.14,7.2and9.5were also obtained from ProteinSimple.Samples of three different IgG2mAb formulations, which are referred to as mAb1,mAb2and mAb3,were prepared by recombinant DNA technology at Pﬁzer Inc.2.2.ChromatographyThe IEX chromatography was performed on a Dionex DX-500liquid chromatograph(Thermo Fisher Scientiﬁc,Lane Cove, Australia)consisting of a GP50Gradient Pump,AD25UV/Vis Absorbance Detector,AS50Thermal Compartment and AS50 Autosampler.Detection was performed at280nm.Flow-rate was 1mL/min,the injection volume was10␮L and the column com-partment temperature was set at30◦C.Instrument control and data acquisition were performed using Dionex Chromeleon soft-ware,version6.80SR5.Chromatograms were transferred to ASCII ﬁles and redrawn using Origin8.1(Northampton,MA).150M.Talebi et al./J.Chromatogr.A1317 (2013) 148–154 The monolithic IEX columns used were ProSwift TM SCX-1S andProSwift TM WCX-1S(4.6mm×50mm)and the packed column wasProPac WCX-10,4mm×250mm,all from Dionex.The monolithiccolumns are methacrylates-based with sulfonic acid and carboxylicacid functionality for SCX and WCX,respectively.The ProPac WCXis a tentacle type ion-exchanger bearing carboxylate groups.Unless otherwise stated,mobile phases were generally preparedby dissolving appropriate amounts of the buffer components inwater prior to splitting into two aliquots denoted as eluent A andB.The pH of each portion was then adjusted with concentratedsodium hydroxide or hydrochloric acid.The elution was performedby a linearly ascending pH gradient from0%to100%eluent B fol-lowed by isocratic elution for3min before returning the eluentcomposition to the starting condition(100%eluent A).The gradi-ent volumes were10and30mL for monolithic and packed columns,corresponding to about14and10column volumes,respectively.For each elution,the column was pre-equilibrated with at leastthree column volumes of eluent A prior to sample introduction.Before measurement of peak areas,each sample chromatogram was subtracted from the relevant blank injection prepared from eluent A.Fractions of the column eluent were collected every1min and the ofﬂine pH measurement was carried out using a pH metre model labCHEM-CP from TPS(Springwood,QLD,Australia).All eluents were prepared using water puriﬁed via a Milli-Q water puriﬁcation system(Millipore,Bedford,MA)andﬁltered through a0.2␮m nylonﬁlter prior to use.mAb samples were ana-lysed as received without buffer exchange or any other sample pretreatment process.After dilution in eluent A to a concentra-tion of approximately0.3mg/mL,samples were stored at5◦C until analysed.2.3.Liquid chromatography–mass spectrometry(LC–MS)CEX chromatography was carried out using a ProSwift TM WCX-1S(4.6mm×50mm)column under pH gradient mode.5mM AMH buffer containing20%(v/v)methanol at pH9.5was used as eluent A and at pH10.5as eluent B.pH of eluents was adjusted before mixing with methanol.Elution was performed by running a linear gradient of eluent A to eluent B in20min at aﬂow-rate of0.4mL/min,which was split(1:100)before introducing into MS.Hyphenated with the CEX chromatography,electrospray ioni-sation time ofﬂight(ESI-TOF)mass spectrometry was performed on a micrOTOF-Q mass spectrometer(Bruker Daltonics,Melbourne, Australia)equipped with an Agilent G1385A microﬂow nebuliser (Agilent technologies,Melbourne,Australia).The instrument was run in a positive ion mode with m/z range of500–10,000and a capillary voltage of4500V(−500V end plate offset).Drying gas ﬂow of5L/min at300◦C was used with a20.3psi nebuliser gas pressure.The instrument was tuned and calibrated using an Agi-lent ES Tuning Mix(catalogue no.G2431A)in enhanced quadratic mode.The deconvolution of ESI mass spectra was performed usinga maximum entropy algorithm(Bruker Daltonics).2.4.Imaged capillary electrophoresis(iCE)iCE proﬁles of mAbs were obtained using an iCE280analyser with operational software from Convergent Bioscience,equipped with an Alcott719AL autosampler.A transparent capillary column (50mm,100␮m i.d.)was used with its inner surface coated with ﬂuorocarbon to minimise electroosmoticﬂow.The test solutions were prepared using various amounts of p I markers,pharmalyte, 1%methyl cellulose,5M urea,20%sucrose,and mAb samples. Throughout the analysis,the capillary was kept at ambient tem-perature while the autosampler was set at8or15◦C,depending on the mAb sample analysed.The injection volume was35␮L and the Fig.1.pH gradient proﬁles obtained for mAb1and mAb2.The mobile phase com-position was12.5mM DEA and12.5mM TEA to either pH7.75(eluent A)or pH 10(eluent B).Gradient:0–100%B in10min,100%B for3min.Column:ProSwift SCX-1S(4.6mm×50mm);Detection:UV at280nm;Flow-rate:1mL/min;Column compartment temperature:30◦C.analysis was performed by applying a sample transfer time of100s, pre-focusing at1500V for duration of1min followed by focus-ing for5min at3kV.Detection was performed at280nm.Further details of the iCE conditions used are provided in the Supporting Information.3.Results and discussionWith the aim of improving the resolution,a series of new buffer systems based on both organic and inorganic buffer species were designed and applied using monolithic columns.To obtain sufﬁ-cient binding of the proteins to the cation-exchanger,the lower pH of the gradient was chosen to be at least1pH unit below the elec-trophoretic p I values of mAbs,that is8.8for mAb1,8.5for mAb2 and8.4for mAb3.3.1.TEA-DEA buffer systemTheﬁrst successful buffer system in eluting two of the mAbs of interest was prepared by mixing equimolar amounts of TEA (p K a7.76)and DEA(p K a8.88)resulting in a system buffering the pH range of approximately7.5–10.Fig.1shows the separation achieved for mAb1and mAb2on a ProSwift SCX-1S column using this buffer system in the pH range of7.75–10with each buffer com-ponent at a concentration of12.5mM.A somewhat linear pH proﬁle for this system over the studied pH range was achieved(Fig.1).No elution was observed for mAb3.Acidic isoforms(p I lower than the main component)are observed for mAb1,while basic isoforms are more pronounced for mAb2.Indications of additional isoforms are also present,but as barely discernible shoulders of the main peaks.The effect ofﬂattening the pH gradient proﬁle on chromato-graphic resolution was of special interest in this study.As the pH gradient slope is reduced there is more time for differential move-ment of the isoforms through the column,which could lead to better resolution[6].In chromatofocusing,it is possible to generate shallow gradient slopes by limiting the pH range of the gradient or reducing the concentration of the mobile phase buffer components [12,15,27].Data presented later in this study show that these two strategies in obtaining higher resolution are also applicable to the external pH gradient approach.M.Talebi et al./J.Chromatogr.A 1317 (2013) 148–154151Fig.2.The effect of eluent concentration (DEA)on the elution proﬁle of mAb2.(A)20mM,pH 9–10;(B)10mM,pH 9–10;(C)5mM,pH 9.2–10.2.For elution to occur at 5mM concentration,more basic pH range is required.Other conditions as in Fig.1.3.2.DEA buffer systemAs seen in Fig.1,elution of mAbs in the TEA-DEA buffer system occurred around the end of the pH range applied.The pH of eluent A was therefore increased from 7.5to 9.A simultaneous reduc-tion in gradient slope was achieved as the gradient time remained unchanged at 10min.In addition,because of its negligible buffer capacity in the new pH range,TEA was removed from the buffer system.The inﬂuence of buffer concentration within the range 20–5mM on separation efﬁciency of mAb2isoforms is shown in Fig.2.A decrease in buffer concentration at the same gradient slope results in an increase in the resolution of the charge variants from the main peak.For elution at 5mM,a further increase in working pH range from 9–10to 9.2–10.2is required.These ﬁndings are in agreement with Farnan and Moreno [9],who achieved improved separation efﬁciency and higher resolution for mAb isoforms by a 4-fold decrease in the concentration of buffer composition.The impact of column chemistry on separation efﬁciency was also evaluated for mAb1(Fig.3)and mAb2(Fig.S1in the Supporting Information).As can be seen,a trivial impact of column chemistry on the selectivity is recognisable.However,there are more promi-nent ﬂuctuations in the pH proﬁle and a longer titration time for the weak cation exchanger (see pH proﬁles).As the workingpHparison of separation of variants for mAb1on ProSwift WCX-1S and ProSwift SCX-1S columns.Eluent:5mM DEA,pH9.2–10.2.Fig.4.Interrelationship between eluent concentration and pH range on separation efﬁciency of mAb1.The gradient slope was 0.1pH units/min.Eluent:5mM AMH,pH 9.2–10.2(A);2.5mM AMH,pH 9.5–10.5(B).range is high enough to ensure full ionisation of the carboxylic group of the weak cation exchanger (p K a ∼5),the reason for dif-ferences in the pH proﬁle might be due to the different chemistries of the stationary phases [25].3.3.AMH buffer systemAlthough suitable for resolving the isoforms of given mAbs,the low volatility of DEA might limit its application for mass spectro-metric detection.In order to address this issue,we explored the use of AMH which is a volatile buffer species with p K a 9.25.For this buffer,acceptable chromatographic resolution of protein isoforms was obtained for even lower concentrations than 5mM (Fig.4).This indicates that the focusing effect of the buffer system increases by decreasing the concentration.Based on earlier results,the optimum pH range had to be adjusted when decreasing the eluent concen-tration to allow maximum separation efﬁciency.Fig.5displays the effect of eluent pH range and gradient slope on resolving mAb1isoforms.By maintaining the gradient slope at 0.1pH units/min,it was found that although the ﬁne structure of the acidicregionFig.5.Inﬂuence of operational pH range and gradient slope on resolution of mAb1variants.Eluent:2.5mM AMH.pH range and gradient slope:9.3–10.3and 0.1(A);9.5–10.5and 0.1(B);9.7–10.5and 0.08pH units/min (C).152M.Talebi et al./J.Chromatogr.A1317 (2013) 148–154 remains unaltered(Fig.5,traces A and B),basic variants previouslyhidden within the threshold of the major peak were clearly resolvedwhen the pH range was raised0.2pH units further from9.3–10.3to9.5–10.5.This step-wise optimisation protocol illustrates the pos-sibilities offered when using a pH gradient over a narrow pH range,in that it enables not only formation of controlled pH proﬁle,butalso permits theﬁne tuning of pH within the range deﬁned by theapplied buffer system to obtain the desired separation efﬁciency.Interestingly,it was found that low ionic strength eluents gen-erated a signiﬁcant back-pressure with the ProPac WCX-10column(pressure upper limit=120bar).Once eluted with5mM AMH pH9.5at0.5mL/min,the initial back-pressure of94bar was monitoredand found to increase gradually.This behaviour is most likely dueto the osmotic pressure generated from the difference between thewater content of the very dilute eluent and the IEX sorbent.Unlikethe packed column,the higher permeability and rigid structure ofmonolithic ion-exchangers resulting from their porous propertiespermits fast generation of pH gradients at moderate and stableback-pressure(<70bar)even at very low buffer concentrations,as well as minimising column titration times(typically less than5min).These merits offer a rapid analysis time that is applicablefor high-throughput process development.While quite successful in resolving charge heterogeneity ofmAb1and mAb2,the simpliﬁed buffer systems failed to elute mAb3unless the eluent ionic strength was increased through addition ofa salt.Rozhkova[7]has previously reported the suitability of con-ducting pH gradient separation of mAb variants by adding NaClinto eluents.Accordingly,2.5mM AMH eluents,pH9–10contain-ing different concentrations of NaCl ranging from20to40mMwere used for eluting mAb3.Results indicate partial resolving ofthe main component from part of the acidic species(Fig.S2inthe SI).Basic variants,however,remained entirely hidden underthe wide shoulder of the major peak.One possible explanation forthis strong retention might be the differences in modiﬁcation site,type of modiﬁcation,and/or degree of modiﬁcation occurring inthe protein[2],all of which inﬂuence the strength of interactionsbetween the protein molecule and the ion-exchanger.These modi-ﬁcations vary from those that change the number of charge residueson the surface of the protein to those being less connected to thecharge but can change antibody conformation.Deamidation,forexample,is one possible modiﬁcation which is likely to have aneffect on retention of a protein by affecting the number of posi-tively charge groups over the surface of a protein and hence itsbinding to a cation-exchanger[3].Further investigation is requiredto conﬁdently determine the characteristics of the mAb variants.3.4.Effects of eluent concentration and pH on resolutionThe overriding consideration in this work was towards maxi-mum achievable resolution for mAb isoforms.pH and ionic strengthare two major characteristics of the eluent governing the elutionand separation of proteins in pH gradient IEX chromatography.Here,we take advantage of the general expressions proposed bySluyterman and Elgersma[14]for the pH gradient approach toexplain the interplay between these two parameters and theireffects on separation efﬁciency.Peak width and peak separation are the two determinants ofresolution.The width of a protein band in terms of pH units can bewritten as:( pH)2≈D(d pH/dV)ϕ(dZ/d pH)(1)where D denotes the diffusion coefﬁcient of a protein,d pH/dV the pH gradient slope andϕis equivalent to the dimensionless Donnan potential[14].This equation implies that an increase in peak focusing is consistent with the lower ionic strength(buffer concentration)used,which increases the absolute value ofϕ.Evi-dence of this inference can be seen in Fig.2,in which the resolution gain for the mAb2main isoform can be related to the focusing effect obtained by decreasing the ionic strength.In fact,the capability of focusing eluent bands is known as one inherent advantage of pH gradient IEX chromatography over conventional salt gradient at aﬁxed pH[18],in which the absence of a focusing effect can be partly related to the lack of the Donnan potential,as a result of the high salt concentration involved.Trace C in Fig.2indicates that while employing the same pH range is likely to maintain the dZ/d pH unchanged,the positive effect of this kinetic factor on peak width can be highlighted by shifting up the pH range further,which along with more decrease in ionic strength leads to an even greater increase in resolution.The dominating effect of dZ/d pH on peak focusing can also be seen by comparing traces A and B in Fig.5 where there is likely no signiﬁcant difference between the Don-nan potentials due to the constant eluent concentration(2.5mM). As should be expected,the peaks became broader when the pH gradient slope(d pH/dV),as another determinant of peak width in Eq.(1),decreased further from0.1(trace B)to0.08pH unit mL−1 (trace C)by keeping the other conditions unchanged,probably due to the domination of another kinetic determinant,i.e.,diffusion coefﬁcient of protein(D).This therefore suggests that the rate of titrating the ion-exchanger with pH has become lower than the equilibrium state of protein molecules,which could compromise the peak focusing gains from shallower gradients.The contribution of the other factor governing resolution,i.e., peak separation,appears to be the main inﬂuence on resolution gains for isoforms in Fig.4,where the peak focussing for main iso-forms seems to be compromised,despite the expected focussing effects as the eluent concentration decreases and the pH range shifts up further.In fact,almost all of the posttranslational mod-iﬁcations and degradations can change surface charge properties of an antibody,either directly by changing the number of charged groups or indirectly by introducing conformational alterations[2]. According to the electrostatic model developed by Tsonev and Hirsh [6]there is a relationship between the magnitude of a shift in electrophoretic p I and the relative charge distribution in a given protein.This,in turn,implies that isoforms can be resolved based on their apparent isoelectric point(p I app,being the pH at which the protein is eluted from the column)[12]when titrating by a gra-dient of pH,relating the resolution achieved in Fig.4to a greater separation of the peaks(for more discussion on p I app and retention mechanism see SI).Similar arguments based on the distribution of charges on the surface of a protein have also been used by other workers to explain the trends observed in resolution for chromato-focusing of␤-lactoglobulin A and B[12],and haemoglobin variants [12,28].3.5.Loading capacityThe loading capacity of the proposed approach for the separa-tion of mAb charge variants was also assessed.While some minor loss of resolution occurred when a sample load of about118␮g mAb1was injected onto the column,the overall separation pat-tern and theﬁne structure of the acidic region remained unaltered (Fig.S3in the SI).By considering the low ionic strength of the buffer system employed,a signiﬁcant potential of this approach for scale up can be seen,enabling it to be used along with classical IEX chromatography for preparative purposes.3.6.Proﬁling charge heterogeneity of mAbs by iCETo assess the resolving power offered by the developed proce-dure,analysis of mAbs by iCE was also included in the study.The difference in separation mechanism of each technique can offer。

重组贻贝粘蛋白的表征及功效评价

生物技术进展 2023 年第 13 卷第 4 期 596 ~ 603Current Biotechnology ISSN 2095‑2341研究论文Articles重组贻贝粘蛋白的表征及功效评价李敏，魏文培，乔莎，郝东，周浩，赵硕文，张立峰，侯增淼 *西安德诺海思医疗科技有限公司，西安 710000摘要：为了推进重组贻贝粘蛋白在医疗、化妆品领域的应用，对大肠杆菌规模化发酵及纯化生产获得的重组贻贝粘蛋白进行了表征及功效评价。

经Edman 降解法、基质辅助激光解吸电离飞行时间质谱、PITC 法、非还原型SDS -聚丙烯酰胺凝胶电泳法、凝胶法、改良的Arnow 法对重组贻贝粘蛋白进行氨基酸N 端测序、相对分子量分析、氨基酸组成分析、蛋白纯度分析、内毒素含量测定、多巴含量测定；通过细胞迁移、斑马鱼尾鳍修复效果对重组贻贝粘蛋白进行功效评价。

结果显示，获得的重组贻贝粘蛋白与理论的一级结构一致，蛋白纯度达95%以上，内毒素<10 EU ·mg -1,多巴含量大于5%；重组贻贝粘蛋白浓度为60 μg ·mL -1时能够显著促进细胞增殖的活性（P <0.01）；斑马鱼尾鳍面积样品组与模型对照组相比极显著增加（P <0.001）。

研究结果表明，重组贻贝粘蛋白具有显著的促细胞迁移和修复愈合的功效，具备作为生物医学材料的潜质。

关键词：贻贝粘蛋白；基因重组；生物材料；表征；功效评价DOI ：10.19586/j.20952341.2023.0021 中图分类号：S985.3+1 文献标志码：ACharacterization and Efficacy Evaluation of Recombinant Mussel Adhesive ProteinLI Min ， WEI Wenpei ， QIAO Sha ， HAO Dong ， ZHOU Hao ， ZHAO Shuowen ， ZHANG Lifeng ，HOU Zengmiao *Xi'an DeNovo Hith Medical Technology Co.， Ltd ， Xi'an 710000， ChinaAbstract ：In order to promote the application of recombinant mussel adhesive protein in the medical and cosmetics field ， the recombi⁃nant mussel adhesive protein obtained from scale fermentation and purification of Escherichia coli was characterized and its efficacy was evaluated. Amino acid N -terminal sequencing ， relative molecular weight analysis ， amino acid composition analysis ， protein purityanalysis ， endotoxin content ， dihydroxyphenylalanine （DOPA ） content of recombinant mussel adhesive protein were determined by the following methods ： Edman degradation ， matrix -assisted laser desorption ionization time -of -flight mass spectrometry （MALDI -TOF -MS ）， phenyl -isothiocyanate （PITC ）， nonreductive SDS -polyacrylamide gel electrophoresis （SDS -PAGE ）， gel method ， modified Ar⁃now. The efficacy of recombinant mussel adhesive protein was evaluated by cell migration and repairing effect of zebrafish tail fin. Re⁃sults showed that the obtained recombinant mussel adhesive protein was confirmed to be consistent with the theoretical primary structure ， protein purity of more than 95%， endotoxin <10 EU ·mg -1， DOPA content above 5%. When the recombinant mussel adhesive protein concentration was 60 μg ·mL -1， the effect of promoting cell proliferation was the most obvious ， and it had very significant activity （P <0.01）. The caudal fin area of zebrafish in sample group was significantly increased compared with model control group （P <0.001）. The results indicated that recombinant mussel adhesive protein can promote cell migration and repair healing and has the potential to be used as biomedical materials.Key words ：mussel adhesive protein ； gene recombination ； biological materials ； representation ； efficacy evaluation贻贝粘蛋白（mussel adhesive protein ， MAP ）也称作贻贝足丝蛋白（mussel foot protein ，Mfps ），收稿日期：2023⁃02⁃24；接受日期：2023⁃03⁃31联系方式：李敏 E -mail:*******************;*通信作者侯增淼 E -mail:***********************.cn李敏，等：重组贻贝粘蛋白的表征及功效评价是海洋贝类——紫贻贝（Mytilus galloprovincalis）、厚壳贻贝（Mytilus coruscus）、翡翠贻贝（Perna viri⁃dis）等分泌的一种特殊的蛋白质，贻贝中含有多种贻贝粘蛋白，包括贻贝粘蛋白（Mfp 1～6）、前胶原蛋白（precollagens）和基质蛋白（matrix proteins）等［1］。

AFM AND XPS STUDIES OF PROTEIN ADSORPTION ONTO GRAPHITE SURFACES

AFM AND XPS STUDIES OF PROTEIN ADSORPTIONONTO GRAPHITE SURFACESAnca Orăşanu and Robert H. BradleyAdvanced Materials & Biomaterials Research Centre, School of Engineering, The Robert Gordon University, Aberdeen AB25 1HG (UK) Corresponding author e-mail address: r.bradley@IntroductionThe literature of Carbon Science contains very little information about the behaviour of carbon surfaces when exposed to physiological environments, such as those which support cell adhesion, even though carbon based materials have been investigated for use in prosthetic devices such as heart valves, ligament repair and vascular stents. The concentration and conformation of adsorbed proteins is critical in determining specific interactions between cell adhesion species (integrins) and biomaterial surfaces in a wide range of situations.In this study, highly orientated pyrolytic graphite (HOPG) has been used as a model surface for the study of the behaviour of two human plasma proteins which is of high biological importance in contact with biomaterials surfaces.ExperimentalHuman serum albumin (HSA) adsorption onto freshly cleaved HOPG surface (basal plane) was studied using atomic force spectroscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Preliminary studies upon human plasma fibronectin (Fn) adsorption onto HOPG were performed using the AFM.Protein solutions of concentration 0.001,0.005, 0.01, 0.05, 0.1, 0.5, 1, 2 and 4mg/ml were prepared using HSA (essentially fatty acid and globulin lyophilized powder, purity 99%) in Dullbeco’s phosphate buffered saline (pH=7.2). Fibronectin solutions of concentration 0.1, 0.5, 1 , 5 and 10 µg/ml were prepared using human plasma fibronectin (Sigma) and trisbuffer saline, pH=7.5, 0.05M. Droplets (200µl) of the protein solution were incubated at 37°C for 1h in contact with the HOPG surfaces, rinsed and then washed in MilliQ water for 4 hours under agitation. The samples were dried slowly at room temperature overnight (12-14h). In order to study the influence of drying conditions on the AFM imaged topography of the adsorbed HSA layer a second set of HSA adsorbed samples was dried relatively quickly under a gentle flow of warm air (37°C) for approximately two minutes.The AFM analyses were performed using a Digital Instruments Nanoscope III, under ambient conditions in tapping mode, immediately after drying of the samples. The surface chemical composition was analyzed using a Kratos X-ray photoelectronspectrometer. Contact angle measurements were performed using a 20µl static drop of distilled water.Results and DiscussionThe substrate used is essentially atomically smooth and the AFM images indicate a RMS roughness of 0.19-0.26nm and contain some small steps of height of approximately 0.3-0.7nm which probably correspond to dislocations of 1 or 2 graphite planes [1].Individual features have been identified when imaging the basal planes after adsorption from an HSA solution of concentration 1µg/ml. These features present two different shapes: one triangular/circular with a diameter of 25±3nm and the other oblong of lengths 35±5nm width 24±2nm and height about 3nm (Figure 1). Due to the tip radius of curvature the true width of the imaged features is extended by 10-20nm. Human serum albumin is known to present different conformations in aqueous solutions as a function of p H. The normal form which is essentially heart shaped with dimensions of 8x8x3nm and the F form, which is oblong and 4x12.9nm [2] could be attributed to the molecular features detected in these experiments (Figures 1a and 1b).a. b.Figure 1.TM AFM topographic images of probably individual HSA molecules showing two shapes: triangular(a),probably N form and oblong (b) possibly F form. The size of the image is:55X55X7nm(a) and 90X90X7nm(b)In both of the drying conditions studied preferred adsorption of the protein molecules along the steps was noticed (Figure 2a) which agrees with data reported by Quist for albumin adsorbed on mica [3] and by Berrie for fibrinogen on graphite [4]. At neutral p H HSA is reported to be 8nm wide and 3 nm height i.e. about 10 to 20 times higher than the step features evident on these surfaces. Interestingly, when the images of the protein adsorbed along the graphite-steps are studied under higher magnification, the protein molecules appear to bridge the step, with one part of the molecule ‘sitting’ on the lower plane and one part on the upper plane. The overall apparent preference for the steps could be due to the presence of oxygen species adsorbed at valence electrons onthe edge of the plane, effectively forming a line of polar sites along the step. There is also likely to be an enhanced dispersion potential along the step which results from the overlapping of the individual potential wells associated with the plane and the adjacent edge.a. b.Figure 2. Topographic AFM images of individual features of proteins adsorbed on HOPG: a.-adsorption from an HSA solution(1µg/ml) imaged after a short time of drying, the surface was washed in PBS prior to water washing in order to remove the weaker bonded proteins and achieve a better image of individual features; b.-adsorption of a Fnsolution (0.1µg/ml) (overnight dried)In the case of fibronectin adsorption from the weakest solution studied (0.1µg/ml) individual features which were either looped, folded or extended have been identified in the AFM images. The extended features are 60-80nm long and 1.2±2nm high as shown in Figure 2b and are very similar in size and shape to those reported by Lin [5] on mica surfaces. Fibronectin is a dimer, formed by two very similar monomers which are joined near their C-termini by two disulfide bonds. Electron microscopy studies reported the Fn molecule to be a long, thin, highly flexible strand with the length of the dimer molecule varying from 120 up to 160nm with a width of 2nm [6]. Therefore, the extended features measured in our study could be assigned to a monomer/half molecule of the fibronectin. Previous biophysical studies suggested that Fn molecules are folded in low salt solutions (2 mM) and extended in higher salt (200mM) and high p H buffers [7,8].a. b .Figure 3.a. Topographic image of a possible dimer of FN with an extended arm and a folded one; b. Top view and cross sections image of an extended monomer (the lengthis 70nm and the height from 1 to 1.2 nm)Most of the imaged features seem to be sharply curved and bent, probably crossing each other to produce a tangled and compact structure. They appear to be similar with the model proposed by Rocco [7] which can be explained by the surface binding process or the fact that the samples were washed with water prior to imaging which will induce the more folded form [7,8].When protein was adsorbed on HOPG surfaces at higher concentrations and dried overnight the formation of a network, with peak and ridges was observed.As the protein solution concentration increases, the protein has a tendency to aggregate into a structure with narrower valleys and wider ridges, thus the network appearing more compact. Similar behaviour was identified in the case of the both HSA and Fn as showed in Figure 4a and 4c.a. b.c. d.Figure 4. TM-AFM images of HSA (a, d) and Fn (b, c) solution adsorbed on HOPG surfaces of concentration 0.01mg/ml(a, b, c) and 2mg/ml(d). The samples were driedovernight(a, c) or for only few minutes(b, d)For the samples dried quickly, a very different type of structure was imaged with no network-type structure apparent and with a grainy type topography (Figure 4b and 4d). The images are very similar at different concentration, the average height being around 1nm, and the RMS roughness in the range 0.5-0.7nm. At higher concentrations some grains of bigger size are apparent which may be due to aggregates of HSA formed in solution prior to adsorption or may be impurities such as globulin present in the HSA.These very different images of the protein film in different drying conditions led us to try another experiment. The protein adsorbed from the weakest HSA solution was imaged in situ, while drying. The initial images show individual features which are randomly arranged on the basal planes. After one hour of drying at room temperature in the AFM apparatus the same surface gave an image which showed a more organized structure with the proteins showing the tendency to arrange into the network structure with circular gaps. After drying overnight the sample was imaged again and now showed a well formed network circular shaped holes. The depth of the holes appears to correspond with the thickness of the film and therefore reveals the substrate. These features are thought to be formed by the de-wetting of the HAS from the hydrophobic HSA surface, there may also be an associated change of protein conformation.The XPS survey spectra of freshly cleaved HOPG show only one single C1s peak at a binding energy of 284.6 eV. After protein adsorption a second peak appears at 400 eV which is the proteinaceous N1s peak. When a blank sample was analyzed (0mgHSA/mlPBS) an O1s peak appears at 530 eV, the oxygen being thought to originate from adsorption of water or oxygen onto the graphite surface or from impurities from the tape used to peel the graphite. The small silicon peaks that appear in the washed samples are also probably due to exposure of the tape (Figure 5). This oxygen and silicon was considered irrelevant in the semi-quantitative assay of the protein adsorption onto the graphite surface.Figure 5. Survey spectra of the samples after the HSA adsorption (left-0mg/ml and right-4mg/ml)The nitrogen atomic concentration has been used as a measure of the irreversibly bound albumin and, as shown in Figure 6, was found to increase with increasing HSA solution concentration. Also shown are average water contact angles (measured after protein was adsorbed) which shows that θ increases slightly as the level of surface protein increases. When the AFM images presented above are analysed to derive roughness data it was found that RMS roughness also increases with adsorbed protein concentration.The XPS, AFM and water contact angle measurements indicate that at lower protein solution concentrations (up to 0.001mg/ml) there is a direct correlation between the amount of adsorbed protein and the protein solution concentration which suggests that the adsorption process is diffusion controlled. After 0.1mg/ml the N1s signal becomes constant at between 3.5 and 3.8 atom % and the contact angle levels out at about 70° which suggests that surface is fully covered with layer of protein. After the point when the layer is complete its surface properties do not change significantly but of course the layer may become thicker as adsorption proceeds.Log 10(concentration)N i t r o g e n A t o m i c P e r c e n t a g e [%]R M S R o u g h n e s s [n m ]Contact angle (degrees) Figure 6. Nitrogen atomic percentage, RMS roughness and average contact anglevariation with the log 10(concentration) of the HSA solutionAs all the data presented in Figure 6 were obtained from the overnight/slow driedsamples a further discussion about these results should be considered. According to the theories of protein adsorption onto solid surfaces [9], as contact or residence time increases, the protein tends to orientationally and conformationally adjust with respect to the interface. In the initial moments after the removal of the solution, protein might still have the same conformation as in the solid-liquid system, i.e. hydrophilic region oriented towards the solution and hydrophobic region towards the hydrophobicsubstrate [10]. When the samples are drying it is expected that the hydrophobic portion of the protein is exposed to air while the hydrophilic portion will be orientated towards aqueous phase. The protein would be expected to adjust its orientation andconformation to the air-solid interface, this being a possible explanation of the very different images obtained for the different drying conditions. Therefore, further AFM studies under a liquid environment would give a better understanding of protein adsorption on HOPG. This, and quantitative measurements of HSA adsorption are being attempted at present.Conclusions/SummaryHSA and Fn adsorption onto HOPG was studied.AFM topographic analysis showed the formation of a network or a grainy structure, dependent upon drying conditions.For adsorption from the HSA solution of concentration 1µg/ml and Fn solution of concentration 0.1µg/ml individual molecules have been imaged. HSA appear to take one of two forms depending upon the adsorption energy and site. Fn appears to be adsorbed into the coiled, bent form.XPS results show an increase of the nitrogen atomic percent of the surface with increasing initial protein solution concentration. Contact angles decrease drastically after protein adsorption then increases slightly up to ~70°.References[1] Marsh H, Science of Carbon Materials. 2000:132.[2] Carter DC, Ho JX. Structure of serum albumin. Advances in Protein Chemistry 1994;45:153-203[3] Quist AP, Bjorck LP, Reimann CT, Oscarsson SO, Sundqvist BUR. A scanning force microscopy study of human serum albumin and porcine pancreas trypsin adsorption on mica surfaces. Surface Science Letters 1995;325:L406-L412[4] Marchin KL, Berrie CL. Conformational changes in the plasma protein fibrinogen upon adsorption to graphite and mica investigated by AFM. Langmuir 2003;19:9883-9888[5] Lin H, Lal R, Clegg DO. Imaging and Mapping Heparin-Binding Sited on Single Fibronectin Molecules with Atomic Force Microscopy. Biochemistry 2000;39:3192-3196 [6] Erickson PH, Carrell N, McDonagh J. Fibronectin molecule visualized in electron microscopy:a long, thin, flexible strand. The Journal of Cell Biology 1981;91:673-678 [7] Rocco M, Carson M, Hantgan R, McDonagh J, Hermans J. Dependence of the shape of the plasma fibronectin molecule on solvent composition. The Journal of Biological Chemistry 1983;258: 14545-14549[8] Erickson PH, Carrell N. Fibronectin in extended and compact conformations. The Journal of Biological Chemistry 1983;258:14539-14544[9] Andrade JD, Hlady V. Protein Adsorption and Materials Biocompatibility:A Tutorial Review and Suggested Hypotheses. Advances in Polymer Science 1986(79):3-63 [10] Browne MM, Lubarsky GV, Davidson MR, Bradley RH. Protein adsorption onto polystyrene surfaces studied by XPS and AFM. Surface Science 2004;553:155-167。

蛋白质在银岛膜上的荧光增强效应及其分析应用.doc

山东大学学士学位论文论文题目：蛋白质在银岛膜上的荧光增强效应及其分析应用作者姓名：李芬妮指导教师：杨景和教授2006年5月25日目录中文摘要 (3)ABSTRACT (4)符号说明 (5)第一章引言 (6)1.1荧光染料探针 (6)1.2稀土离子及其螯合物探针 (7)1.3荧光衍生化试剂 (8)1.4近红外染料探针 (8)1.5纳米粒子荧光探针 (8)1.6纳米粒子应用于检测生物分子 (10)第二章实验部分 (13)2.1 试剂 (13)2.2 仪器 (13)2.3 实验步骤 (13)2.4 结果与讨论 (17)2.4.1 扫描电镜图 (17)2.4.2 荧光光谱 (18)2.4.3 胶原蛋白浓度的选择 (19)2.4.4 银岛膜厚度的选择 (20)2.4.5 稳定性实验 (21)2.5 分析应用 (21)2.6 体系反应机理探讨 (21)2.7 结论 (22)参考文献 (24)致谢 (26)附录 (27)摘要蛋白质是生物体的基本组成成分之一，建立蛋白质快速、灵敏、而简便的分析方法，对在分子水平上阐明生命的奥秘及寻找疑难疾病的解决方案等方面具有重要的意义，是当前生物分析化学研究的前沿和热点。

本文利用荧光法研究了银纳米粒子对蛋白质体系的荧光增强效应。

利用沉积法在石英片上制备了银岛膜，研究了银岛膜的厚度和隔离层（胶原蛋白）的厚度、浓度和吸附时间对体系荧光增强的影响。

实验表明，银岛膜厚度在230-280 m 之间时对蛋白质的荧光强度具有最大的增强效应, 在隔离层（胶原蛋白）的存在下，蛋白质体系的荧光增强更大。

通过检测表明在最佳实验条件下，荧光增强的程度与蛋白质的浓度成线性关系,BSA 和HSA的线性范围分别为1×10-7-4×10-5g/ml和1×10-7-7×10-5g/ml,他们的检出限（S/N=3）分别为3.6×10-9g/ml和3.6×10-8g/ml，因而提出了灵敏而简便的蛋白质测定方法。

GⅠGⅡ型诺如病毒两联装甲RNA标准样品的研制

GI/GII型诺如病毒两联装甲RNA标准样品的研制王鸣秋1，2，杨俊2，常雨桐2，张涛1，刘丽娟2（1.湖北省食品质量安全监督检验研究院，湖北武汉 430075）（2.中国检验检疫科学研究院，北京 100176）摘要：针对目前缺乏适配多项检测标准、稳定、安全的诺如病毒RNA标准样品的问题，研制基于MS2噬菌体内含常见GI/GII 型诺如病毒检测靶标两联装甲RNA标准参考样品。

人工合成MS2噬菌体成熟酶基因、衣壳蛋白基因、包装位点及GI/GII型诺如病毒靶标基因，克隆于表达载体pET-28a(+)中，构建重组质粒pET-MS2-NoV。

经大肠杆菌BL21诱导表达，先后利用PEG6000、酶处理和丙烯葡聚糖凝胶层析柱纯化表达产物。

SDS-PAGE和透射电镜鉴定产物大小及结构，荧光定量PCR检测有无残留核酸。

之后对纯化的病毒样颗粒（Virus-like particles，VLPs）开展定值、均匀性和短期稳定性研究。

SDS-PAGE结果表明重组质粒在BL21中表达出了目的蛋白，大小在10~15 ku之间，与预期一致；纯化后的VLPs无杂蛋白和残留核酸；透射电镜下呈结构完整、大小均一的球状，直径约25 nm。

纯化后AR-NoV中GI型和GII型靶标定值结果分别为(4.04±0.62)×107 copies/μL和(6.16±0.30)×107 copies/μL。

单因素方差检验证实样品均一性良好，F<F0.05（25,52）；短期稳定性研究结果表明AR-NoV在37 ℃至少可稳定保存15 d，25 ℃至少稳定保存24 d。

本研究制备的诺如病毒GI/GII型两联装甲RNA标准样品稳定均一，拷贝数高，能够为GI/GII型诺如病毒核酸分子检测提供全过程质控。

关键词：诺如病毒；两联装甲RNA；MS2噬菌体；标准样品；实时荧光定量RT-PCR文章篇号：1673-9078(2021)03-286-293 DOI: 10.13982/j.mfst.1673-9078.2021.3.0835 Preparation of Coupled Armored RNA Reference Material for NorovirusGI/GIIW ANG Ming-qiu1,2, YANG Jun2, CHANG Yu-tong2, ZHANG Tao1, LIU Li-juan2(1.Hubei Provincial Institute for Food Supervision and Test, Wuhan 430075, China)(2.Chinese Academy of Inspection and Quarantine, Beijing 100176, China)Abstract: To provide a safe and stable reference material for Norovirus nucleic detection, the armored RNA containing target RNA of Norovirus GI and GII based on MS2 bacteriophage was developed in this work. DNA fragments including maturase coding gene, capsid protein coding gene and packing site of MS2 bacteriophage, target cDNA sequence of Norovirus GI and GII were synthesized artificially and then cloned into expression vector pET-28a(+) to construct recombinant plasmid pET-MS2-NoV. After expressed in E. coli BL21 cells by IPTG induction, the expression product was purified by PEG6000, enzyme digestion and molecular sieve chromatography. The purified product, also named AR-NoV, was identified by SDS-PAGE, transmission electron microscopy (TEM) and RT-PCR. The value, homogeneity and stability of the AR-NoV were evaluated. SDS-PAGE analysis showed that with the molecular weight of tar-get protein expressed in BL21 was 10~15 ku, which was consistent with the predicted value. There were no impure proteins and residual nucleic acids in AR-NoV after purification. The AR-NoV presented as spherical VLPs with uniform particle size (about 25 nm) and integrated structure under TEM. The values of GI and GII targets in AR-NoV were (4.04±0.62)×107 copies/μL and (6.16±0.30)×107 copies/μL, respectively. The good homogeneity of AR-NoV was confirmed by single-factor ANOVA test (F<F0.05(25,52)). In addition, the stability result indicated that the AR-NoV could be stable at 37 ℃ for 引文格式：王鸣秋,杨俊,常雨桐,等.GI/GII型诺如病毒两联装甲RNA标准样品的研制[J].现代食品科技,2021,37(3):286-293W ANG Ming-qiu, Y ANG Jun, CHANG Y u-tong, et al. Preparation of coupled armored RNA reference material for Norovirus GI/GII [J]. Modern Food Science and Technology, 2021, 37(3): 286-293收稿日期：2020-09-04基金项目：湖北省重点研发计划项目（2020BCA091)；中国检验检疫科学研究院基本科研业务费项目（2019JK017）作者简介：王鸣秋（1986-），女，高级工程师，研究方向：食品微生物检测通讯作者：刘丽娟（1971-），女，博士，研究员，研究方向：病原微生物检测与检疫研究15 days and 25 ℃ for 24 days at least. In conclusion, the armored RNA containing coupled Norovirus GI/GII prepared in this work was stable and uniform, with high copy number, which could help the whole process quality control of molecular detection for Norovirus.Key words: Norovirus; coupled armored RNA; MS2 bacteriophage; reference material; real-time RT-PCR诺如病毒（Norovirus，NoV）属于杯状病毒科（Caliciviridae），为单股正链无包膜RNA病毒，直径约为27~40 nm，全长7.5~7.7 kb。

学经典~威廉姆斯13版l骨质疏松和骨生物学l02-骨重塑及其调控

学经典~威廉姆斯13版l骨质疏松和骨生物学l02-骨重塑及其调控**CK'sEndocrine Notes骨质疏松和骨骼生物学(Osteoporosis and Bone Biology)威廉姆斯内分泌学第13版Chapter 29, Section VII要点：•根据最近的发现，骨骼的功能意义已经得到了彻底的重新挖掘。

例如，除了被认为是骨骼的经典角色的机械支持和矿物储存之外，矿化的间充质组织还输出对循环磷酸盐、全身能量代谢和胰岛素敏感性的调节至关重要的肽。

因此，我们现在对骨骼及其在维持矿物质和代谢体内平衡中的作用有了更完整的了解。

•骨质疏松症是最常见的骨代谢疾病，对生活质量和死亡率有很大影响，因为它对骨强度有负面影响。

在过去的30年里，骨折的危险因素已经得到了详尽的研究。

与此同时，骨重塑的生物化学标记和分别用于评估骨代谢和结构的放射检查现在被用于骨折易感性的早期识别。

•由于我们对导致骨质流失的机制的认识迅速提高，用于预防骨折的成本效益高的药物的开发已经加快。

此外，正在进行的研究指出了更多更有效的治疗骨质疏松症的药物。

•本章重点介绍骨重塑单位的代谢方面及其对激素、遗传和环境变化的反应，以及其对组织体内平衡的重要性。

与其他慢性疾病的治疗方案不同，骨质疏松症治疗处于独特的地位，每周、每月、每半年甚至每年给药可能足以完成成功的治疗。

骨质疏松症医学的当前挑战是定义那些有早期骨骼衰竭风险的女性(有时是男性)。

目录：（本部分已标红）•历史背景•骨骼生物学•骨重塑及其调控•骨质疏松症和骨折的流行病学•骨质疏松症的发病机制•骨质疏松症的治疗方法Chen Kang CK医学科普2019.10第二部分骨重塑及调控内容：•重塑概述•重塑的局部调节•全身激素和骨重塑重塑概述成人骨量由两个过程决定:青春期获得峰值骨量和成年后的骨丢失。

骨量的变化源于骨重塑周期中的生理和病理生理过程，最终可导致骨骼脆弱。

这方面女性最容易受伤害的时期是加速线性生长的青春期(10-16岁)和生命后期(通常是绝经后不久(45-60岁))。

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Pakistan Journal of Nutrition 10 (3): 296-301, 2011ISSN 1680-5194© Asian Network for Scientific Information, 2011296Whey Protein Films and Coatings: A ReviewHossein JooyandehRamin Agricultural and Natural Resources University, Ahvaz (Mollasani), IranAbstract: Whey is a by-product of the cheese-making industry. The most important constituent of whey is Whey Protein (WP) generally used in infant formulas and sports food. Nowadays, great efforts are being made to find out new WPs applications, e.g. production of edible films. Edible or biodegradable films constitute a convenient means to prolong the shelf life of foods and increase their quality without contributing to environmental pollution. Apart from acting as selective barriers for moisture and gas migration, these films may operate as carriers of many functional ingredients. Such ingredients may include antioxidants,antimicrobial agents, flavors, spices and colorants which improve the functionality of the packaging materials by adding novel or extra functions. In this article, the functional properties of edible films made with WPs and their applications in food industry will be reviewed.Key words: Whey protein, edible film, coating, functionality, permeabilityINTRODUCTIONEdible films and coatings have been involved in food preservation for centuries. Since the twelfth century,civilizations have been using wax coatings as a method to lengthen shelf life of foods (Debeaufort et al ., 1998).The main purpose was to prevent the loss of moisture and to maintain quality and texture during storage.Today, waxes are still commonly used to preserve fruits,vegetables and meats for extended shipping and shelf life, but there are now more materials available for edible films and coatings. These alternative sources,along with improved processing techniques, have extended the use of edible films and coatings beyond simple moisture barriers. Since the early 1900s, food-grade shellac resins have been used for improving the appearance of foods (Beckett, 2000; Valencia-Chamorro et al ., 2009). While these particular edible coatings have provided nominal protection or improvement to the physical appearance of foods, their uses are limited.Research in the area of edible films has improved the technology and created more options. Carbohydrates and proteins are biopolymers that can form films like waxes, lipids, or resins. Corn zein has been employed commercially as a food coating since World War II to enhance food appearance (Lawton, 2002). The diverse structures and chemistries of carbohydrates and proteins offer a wide array of films and coating properties. Protein-and carbohydrate-based edible films are generally more cohesive and flexible than wax films and possess better gas-barrier properties at certain conditions. Biopolymers also have the advantage of having more possibilities for adjusting film properties for specific application development through chemistry and processing.Many publications have been written reviewing the properties of edible films and coatings formed from biopolymers such as wheat gluten, soy protein, starch,cellulose and casein (Gennadios and Weller, 1990;Brandenberg et al ., 1993; Krochta, 2002; Gennadios,2002; Zhang and Mittal, 2010). While many biopolymers have been studied as edible films and coatings, this article focuses on Whey Protein (WP).Edible films and coatings: Specifically, an edible film is a thin continuous sheet formed from a biopolymer matrix that is cohesive enough and has the physical integrity to stand alone. The thickness of an edible film is typically 2-10 mils (0.050-0.250 mm). Depending on their thermal properties and surface chemistry, edible films can be formed into pouches or laminated onto other packaging substrates. The main purpose for edible films from biopolymers is to control mass transfer of multiple compounds including gas, aroma, oil and water vapor into or out of a food, preserving food quality. Edible films must also be both strong and flexible to withstand forces experienced during handling and processing. Edible coatings are edible films formed directly on the surface of a food or material. They are typically thinner than stand-alone edible films. While edible coatings themselves can improve the physical integrity of a coated product, they do not necessarily need to be as tough and resilient as a stand-alone film because of the underlying support of food. Additionally, edible coatings can improve appearance of a product by adding color or gloss, making it more appealing to consumers. Edible films are the form used to study mechanical, barrier and surface properties. Coatings are studied as one type of application of an edible film.Most edible films are formed by removing the solvent,formation. Noncovalent aggregation also occurs through called solvent casting. For years, solvent casting has new hydrophobic, ionic and van der Waals interactions been the main method of forming WP films for research.that occur between newly exposed groups of the heat-Two possible solvent choices to keep the films safe for denatured whey protein. These interactions increase as Food-grade are the water and ethanol consumption.pH decreases toward the isoelectric point of WP Solvent casting starts with a dilute solution of(Kinsella, 1984; Kinsella and Whitehead, 1989). biopolymer. The solution is spread in a thin layer on a There are other methods for inducing protein chain level surface and the solvent evaporates to form a film.cross-linking besides heat denaturation. Irradiation has Under ambient conditions, edible film drying can occur been successfully used to cross-link casein proteins, as with hot air, infrared energy, or microwave energy.well as soy proteins (Brault et al., 1997; Lacroix et al., Method of drying can significantly affects the physical2002). A hypothesized mechanism is radical properties of the final film. There are two periods during polymerization through tyrosine and the formation of drying of the films: The Constant Rate Period (CRP) and bityrosine linkages between protein chains. However, the Falling Rate Period (FRP) (Kozempel et al., 2003).WPs are low in tyrosine residues and irradiation alone During the CRP, the major phenomenon is mass does not produce a significant increase in molecular transfer between the surface of the film and air. Once the weight of WP (Vachon et al., 2000).surface comes into equilibrium with air conditions, theFRP begins. During this period of drying, the mass Properties of solvent-cast WP films: The most transfer of water from the film to the air is limited by important characteristics of edible films are mechanical, diffusion of water from the inside of the film to the barrier and appearance properties, because they surface. Some factors are important in air drying are (1)determine under what conditions they can be applied exposed surface area, (2) drying air temperature, (3)and used. As with traditional plastic film packaging, the drying air Relative Humidity (RH), (4) drying air velocity most significant mechanical properties of interest are and (5) drying period (Alcantara et al., 1998). Microwave tensile strength, elastic modulus and percent energy can penetrate more quickly into films, leading to elongation. The most important barrier properties are faster diffusion of water from the center of the film to the determined as film oxygen permeability and water vapor film surface. Different drying conditions can change permeability (Zhang and Mittal, 2010). Carbon dioxide, edible film morphology, which affects appearance,oil and aroma permeability properties are also of barrier and mechanical properties (Perez-Gago and interest, but the information is of value for more specific Krochta, 2000).applications. The most important appearance propertiesare transparency, color and gloss.Whey Protein (WP) film and coating formationWhey Proteins (WPs): There are several individual Mechanical properties: Tensile properties-tensile proteins within the mixture of WP, with $-lactoglobulin, "-strength, elastic modulus, percent elongation and lactalbumin, Bovine Serum Albumin (BSA) and resiliency-are indicators of protein-protein interactions in immunoglobins being the main proteins (deWit and WP film matrices. Tensile strength is the maximum Klarenbeek, 1983; Kinsella, 1984). Among them, the amount of force applied to a film per unit original cross-most abundant and important protein for film formation sectional area before film breakage. Elongation is the is $-lactoglobulin and the second most abundant WP is distance the film will stretch before breaking divided by "-Lactalbumin. the original film length. Resiliency is the film’s overalltoughness. It can be estimated by multiplying tensile Solvent casting of WP films: Solvent-cast WP films, for strength by percent elongation. The tensile properties which the solvent is water, can form from native proteins can be adjusted to make more flexible, stretchable, through the electrostatic interactions, hydrogen bonding resilient films by changing the state of the protein or by and van der Waals forces that occur between the protein the addition of plasticizers. Increased cross-linking that chains as the water evaporates. Native films are occurs during denaturation leads to stronger and stiffer cohesive, but the protein film network can be improved films with greater elongation (Perez-Gago and Krochta, and the resulting solvent-cast film tensile and barrier1999) compared to films made with WP in the native properties improved through heat denaturation and form. The cross-linking of WP chains produces stronger cross-linking of the WP chains (Perez-Gago and Krochta,films, but also allows for greater deformation of the 1999). Thus, most research on WP films has involved films. The amount and type of plasticizer in a WP film heat denaturing of the WP in aqueous solvent and then also affects tensile properties. Plasticizer efficiency, or casting the solution to form a film with cross-linked WP how well a plasticizer adjusts tensile properties, is upon evaporation of the water. Polymerization is not the dependent on the size, shape and compatibility of the only chemical reaction involved in WP film network plasticizer with the protein.297Permeability properties and waxes can greatly improve the water vapor Oxygen permeability: Good oxygen-barrier properties of permeability of WP films, their affect on tensile packaging materials are critical for maintaining the initial properties must be considered. At high levels, especially high quality of the packaged product. The oxidation of for brittle waxes, tensile strength and elongation fats, oils and other food components produces off-decrease and films become brittle and hard to handle flavors, off-colors and nutrient loss. Thus, the protection without breaking (Shellhammer and Krochta, 1997). of the content against oxygen is one of the most However, there is a positive effect on tensile properties important requirements in packaging of food products.of decreasing particle size of insoluble additives in Despite the availability of a variety of excellent synthetic protein films (Dangaran et al., 2006). Perez-Gago and oxygen barriers, the disadvantages of any such Krochta (2001) found tensile strength and elongation composite polymeric structures are the difficulties significantly increased when particle size of beeswax in entailed in their recycling. Existing composite films WP films decreased. Compare to synthetics, WP films containing layers of different plastic materials may not are only moderate moisture barriers. Even with the be recycled, because typically only single component inclusion of lipids, WP films still have higher water vapor plastics are recyclable. Therefore, there is an increasing permeabilities than Low-Density Polyethylene (LDPE), interest in the development of biodegradable polymers High-Density Polyethylene (HDPE) and nylon. In terms of (i.e., biopolymers) for packaging materials that have applications, WP films may be best for food products suitable application properties and can be disposed of needing a low to moderate moisture barrier to avoid after use in an economically and ecologically acceptable condensation from forming on the surface. Moreover, the way. Properties, as well as potential practical uses of appearance needs to be considered because including biopolymer films and coatings based on lipids and waxes confers some opaqueness. They may polysaccharides, proteins and lipids from numerous have some short-term use inside food as protective plant and animal sources, have been well reviewed layers between high and low water activity layers like (Debeaufort et al., 1998; Gennadios, 2002). Particularly cookies and cream fillings or piecrusts and fruit fillings. among biopolymers, the extremely low oxygenpermeability of WP films, in addition to good gloss and Aroma and oil permeability: WP films have been found mechanical properties, makes WP potentially useful as to be excellent barriers to aroma compounds and oil a transparent coating material for improving the oxygen-(Miller et al., 1998). This is consistent with the findings barrier property of food packaging (Miller et al., 1998).that WP limits the flavor perception of benzaldehyde, The compositions of WPI and WPC products also differ citral and D-limonene (Hansen and Heinis, 1992) and in the levels of the various constituents, especially that $-lactoglobulin has been found to be a binder of lactose that acts as a plasticizer. These composition aromatic compounds (Farrell et al., 1987). Miller et al. differences may influence markedly the barrier and(1998) developed a method for determining permeability mechanical properties of WP films. Polyethylene (PE)of aroma compounds through films. They used the and Polypropylene (PP) have been widely used in method to determine that WP films had better barrier to diverse packaging applications due to abundant supply,D-limonene than vinylidene chloride copolymer (co-VDC) low cost, good processability, low energy demand for by 250-15,000 times, depending on relative humidity, but processing and resistance to chemicals and harsh not as good a barrier as Ethylene Vinyl Alcohol environments. Common polyolefin films such as PE and Copolymer (EVOH). By monitoring the penetration of PP are also excellent moisture barriers, but they must be dyed vegetable oil on whey protein-coated paper over coated or laminated with synthetic polymer layers time, Lin and Krochta (2003) compared WP coatings including EVOH (ethylene vinyl alcohol) copolymers,with various plasticizers as barriers to oil. They found PVDC (poly-vinylidene chloride) and nylon to provide an WP plasticized with glycerol (1.3 M) prevented the oxygen barrier (Hernandez et al., 2000). The resulting penetration of oil into the paper for at least 16 h. structures are quite expensive and non-recyclable.Optical properties (color, gloss, haze and transparency): Replacing these synthetic oxygen-barrier layers with WP WP films have the two characteristics-transparent and coatings could provide a new path for use of WP sand highly glossy-that are very important to coating perhaps improve recyclability of the base plastic film applications. Trezza and Krochta (2000) found whey (Hong and Krochta, 2003, 2004). protein-glycerol films had gloss value 90.8. The gloss ofWP films can be affected by plasticizer choice. Lee et al. Water vapor permeability: Since whey is hydrophilic(2002) found sucrose-plasticized WP coatings had the protein, these films are only moderate barriers to highest gloss compared to glycerol-or propylene glycol-moisture at best. The water vapor permeability of WP plasticized coatings. They hypothesized that the films is high, but the barrier properties of the films have Refractive Index (RI) of the plasticizer affected the gloss been improved through addition of hydrophobic of the final film. Amorphous sucrose has an RI higher materials like waxes and lipids. While addition of lipids than other commonly used plasticizers. The amount of298light a surface reflects is related to the RI-the higher the finished dried or confectionery products. Lee et al. RI, the more the light is reflected. Dangaran and Krochta(2002) applied the WPI films as coatings to panned (2003) showed that as sucrose content increased, the chocolate candies and found sucrose-plasticized WPI gloss of WP films and coatings significantly increased.coatings to be the glossiest. However, in a consumer However, crystallization of the sucrose that occurred over study, the lower level of WPI gloss coatings was time gave the WP films a hazy appearance and lowered preferred overall.the gloss. To be acceptable coatings, crystallization of WP films incorporating organic acids and various the plasticizer needed to be controlled. Dangaran and bioactive peptides have been tested against both Krochta (2006a) found that sucrose crystallization in WP spoilage and pathogenic organisms. WP films films could be hindered by the addition of inhibitors.containing antimicrobials provide another layer of Raffinose and modified starch prevented crystal growth protection while potentially reducing the amount of in whey protein-sucrose films for at least 1,800 h of antimicrobials needed for efficacy. The two issues for storage at 53% relative humidity. WP films without active edible films containing antimicrobials are the inhibitors had noticeable crystallization after 50 h of minimum inhibitory concentrations against different storage. When applied as coatings to chocolates for a levels of contamination from specific microorganisms glossy finish, the whey protein-sucrose coatings with and diffusion constants of the antimicrobials in the film raffinose inhibitors maintained gloss longer than WP and in the food. Both have been investigated for WP coatings containing only sucrose (Dangaran and films. Perhaps the greatest potential for active edible Krochta 2006b).films concerns food safety. Chemical antimicrobialcompounds have been and continue to be used. Applications of whey coatings: Based on their inherent However, there is a growing concern about the use of properties, some specific applications of WP films synthetic pesticides and chemicals with foods (Sloan, formed as coatings have been researched and2001). Natural antimicrobials have been researched as developed. By taking advantage of the passive gas effective and socially acceptable alternatives. The barrier, glossy appearance properties or active film bioactive proteins lysozyme, lactoferrin and capabilities, WP films and coatings have been designed lactoperoxidase have been extensively investigated as to be coatings that lengthen shelf life, improve consumer antimicrobials in WP films. As an added layer of acceptability, or raise the level of food safety for products protection, WP films can be used in ready-to-eat meat like nuts, eggs, confectionary, meats and fruits and products such as roasted turkey, smoked salmon and vegetables.sausage products. These products are susceptible to Since nuts and peanuts are susceptible to lipid oxidation contamination during slicing and packaging (Cagri et al., and quickly oxidized when exposed to oxygen and forms2002; Min et al., 2005a). WP coatings with active rancid off-notes, which make the product unacceptable antimicrobials have been shown to be effective at to consumers and shortens shelf life, an excellent inhibiting growth of Escherichia coli O157: H7, oxygen-barrier coating is needed. A WP coating can Salmonella enterica or Listeria monocytogenes, thus apply directly to the nut surface and allow the protective increasing food safety and extending product shelf life layer to remain with the food, also reducing the high-(Min et al., 2005a,b). Enzymes may be also used for performance barrier requirement for the outside product changes in functionality and flavour modification packaging. In a study by Mate et al. (1996), peanuts(Jooyandeh et al., 2009a).coated with WP isolate had lower peroxide and hexanal Nowadays Fresh cut fruits and vegetables are growing formation during storage as compared to uncoated in popularity; however, once cut, the produce becomes peanuts. Krochta (2002) also found that the WP coatings highly perishable. The respiration rate of fresh cut fruitsocould extend the shelf life of peanuts to 273 days at 25C can be 1.2-7 times higher than unprocessed fruit, compared to 136 days for uncoated nuts. Lin and according to Lee et al. (2002). Modified atmosphere Krochta (2003) found that incorporation of surfactant in packaging could slow oxidation, but if oxygen levels are the WP coating significantly increased its coating reduced too low, anaerobic conditions could be created efficiency. and this creates the risk of anaerobic bacterial growth. Whey films also improve shelf life of eggs by reducing Edible coatings that have moderate oxygen, carbon loss of water and carbon dioxide through the shell dioxide and water vapor permeability can be applied to during storage. In a study by Caner (2005), the shelf life the surface of fresh cut product to extend shelf life by of whey protein-coated eggs was 1 week longer and delaying ripening and browning, reducing water and color, yolk index (yolk height and yolk width) and pH aroma loss, carrying antioxidants, or/and carrying texture changed slower and remained at higher quality levels enhancer (Olivas and Barbosa-Canovas, 2005).than uncoated eggs. Because of their inherent characteristics, WP films are As edible films are so glossy and transparent, they can excellent oxygen, aroma and oil barriers without be used to impart a smooth and glossy appearance in adjustment. They can be passive barriers and add a299300layer of protection to foods by being incorporated into the opportunities for change in chemical structure and thus,product as a film layer or a coating. They serve parallel future property improvement than carbohydrates. More functions to traditional packaging materials. They can be applied to traditional packaging materials like paper and plastic films to impart a new functional property. Paper is the most widely used packaging material because of its versatility, printability and easy recyclability. However,since it is made from cellulose, which is hydrophilic,paper is a poor water vapor barrier. Moreover, paper loses its strength and integrity when wet. It is often coated with wax or polyethylene to improve the moisture-barrier properties. A next step in both edible film and traditional packaging technology is the incorporation of functional compounds that confer another protective action to the system creating what is known as active packaging. By definition, active packaging interacts directly with the food or headspace of the product (Han,2000). In traditional packaging systems, the active compounds may be toxic and therefore cannot touch the food directly. To prevent contamination, the active compounds may be incorporated into complex multilayer packaging. As stated previously, layered packaging is difficult to recycle and most often ends up as waste in landfills. Edible films and coatings can also be active layers, but have the benefit of being nontoxic concerning contact with food. WP films can carry such bioactive compounds as flavors, natural oxygen scavengers and antimicrobials without the concern of toxicity.Future trends: Perhaps the biggest story in the dairy industry in the past couple of decades has been the rise of new applications for whey and whey proteins. Once considered a waste product in the cheese manufacturing process, whey and WP products today are used for a wide range of functional and nutritional properties (Jooyandeh, 2009). Applications of WPs in bread (Jooyandeh et al ., 2009b) and cheese (Jooyandeh and Minhas, 2009) to enhance product quality are examples of these properties. An active area of current researches is utilizing WPs to formulate edible and biodegradable films. There is an increasing need for packaging materials that are alternatives to petroleum-based sources. As oil prices continue to go up, so do packaging costs. Renewable sources of materials for packaging will create a steady, reliable supply. 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