Codon optimization, expression and characterization of an internalizing anti-ErbB2 single
chain antibody in Pichia pastoris
Siyi Hu, Liangwei Li, Jingjuan Qiao, Yujie Guo, Liansheng Cheng, Jing Liu?
Lab of Molecular and Cellular Immunology, School of Life Sciences, University of Science &
Technology of China, Hefei, Anhui, 230027, P. R. China
Abstract
Anti-ErbB2 antibodies are used as convenient tools in exploration of ErbB2 functional mechanisms and in treatment of ErbB2-overexpressing tumors. When we employed the yeast P. pastoris to express an anti-ErbB2 single chain antibody (scFv) derived from the tumor-inhibitory monoclonal antibody A21, the yield did not exceed 1-2 mg/L in shake flask cultures. As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and scFv production, we designed and synthesized the full-length scFv gene by choosing the P. pastoris preferred codons while keeping the G+C content at relatively low level. Codon optimization increased the scFv expression level 3-5 fold and up to 6-10 mg/L. Northern blotting further confirmed that the increase of scFv expression was mainly due to the enhancement of translation efficiency. Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5-7.0 with 2% casamino acids after 72h methanol induction. Secreted scFv was easily purified (>95% homogeneous product) from culture supernatants in one-step by using Ni2+ chelating affinity chromatography. The yield was approximately 10-15 mg/L. Functional studies showed that the A21 scFv could be internalized with high efficiency after binding to the ErbB2-overexpressing cells, suggesting this regent may prove especially useful for ErbB2-targeted immunotherapy.
Key Words：ErbB2; scFv; P. pastoris; Codon optimization; Internalization
?Corresponding author: Tel., 86-551-3601437; Fax., 86-551-3601443; E-mail address: jliu@https://www.doczj.com/doc/3914770170.html,
The ErbB2 receptor tyrosine kinase (Her2 / P185) plays an important role in normal cell growth and differentiation as well as in human tumorigenesis [1]. A number of monoclonal antibodies (mAbs) have been developed against the extracellular domain (ECD) of ErbB2, and they are used as convenient tools in exploration of the mechanisms of ErbB2 mediated signal transduction and in treatment of ErbB2-overexpressing tumors [2, 3]. In contrast to the intact antibodies, single chain antibody fragments (scFv) consisting of the variable domains of heavy chain (V H) and light chain (V L) are more suitable for antibody-based immunotherapy [4]. Recently, anti-ErbB2 scFvs which are selected from phage libraries and can be rapidly internalized have been developed as newer tumor targeting molecules [5, 6].
In the previous study, we generated an anti-ErbB2 ECD mAb A21, and it was shown to specifically inhibit the growth of Erbb2-overexpressing cancer cells both in vitro and in vivo [7, 8]. Subsequently, we constructed a single chain chimeric antibody by fusing the V L and V H domains of A21 mAb to human IgG1 Fc. The engineered antibody was expressed in CHO-GS system to evaluate its potential applications in therapy of ErbB2-overexpressing tumors [8, 9]. We also cloned the A21 scFv gene into the pCANTAB5E vector and expressed it in E.coli, but the soluble periplasmic expression was found to be very low (<0.1 mg/L). This did not meet the requirement of large amounts of scFv for studying its biological effects and therapeutic applications.
The yeast Pichia pastoris has been explored to successfully express recombinant antibody fragments with yields typically above several mg/L using the S. cerevisiaeα-factor secretion signal [10-15]. Therefore, we cloned the A21 scFv gene into the pPIC9K vector and employed P. pastoris to express the functional scFv, but the yield did not exceed 1-2 mg/L in the standard shake flask cultures despite the observed high transcription level. Although the expressing capacity of a heterologous protein in P. pastoris is greatly influenced by its inherent properties, the yield can still be significantly enhanced by genetic manipulation of the rate-limiting factors involving gene transcription, mRNA translation, protein folding and secreting [16-17]. As the native A21 scFv gene exhibited a poor codon usage bias for P. pastoris, we considered that it should be one of the limiting factors leading to the inefficient translation and scFv production.
In this paper, we designed and constructed a full-length synthetic gene by changing the codon usage to that preferred by P. pastoris in order to improve the scFv expression. We also investigated the effects of gene copies and culture conditions on scFv accumulation. Under optimal conditions, the scFv expression was increased up to 15 mg/L in shake flask cultures. Further studies showed that the scFv could be rapidly internalized into the cytoplasm after binding to the ErbB2-overexpressing cells, making it an attractive targeting vehicle for antibody-based immunotherapy.
Materials and Methods
Yeast strain and culture medium
The P. pastoris strain GS115, the expression vectors pPIC9 and PPIC9K were purchased from Invitrogen. MD / MM medium (1.34% yeast nitrogen base, 400 ug/L biotin, 2% agar, 2% dextrose or 1% methanol) was used for selecting transformants with Mut+ or Muts methanol utilization phenotype. YPD-G418 medium (1% yeast extract, 2% peptone, 2% dextrose, 2% agar, 0.5-3.0 mg/ml G418) were used for selecting multicopy transformants. The P. pastoris cells were cultured in BMGY medium (1% yeast extract, 2% peptone, 1% glycol, 400ug/L biotin, 0.1 M potassium phosphate, pH 6.0) for growth and in BMMY medium (1% yeast extract, 2% peptone, 400 ug/L
biotin, 1% methanol, 0.1 M potassium phosphate, pH 6.0) for induction.
Antibody and cell line
A21 mAb was purified from ascites as described previously [7]. Human breast cancer SKBR3 cells were cultured in Dullbecco’s modified Eagle’s medium (Gibco BRL) supplemented with 10% fetal bovine serum at 37°C with 5% CO2.
Design of the synA21 gene
The synthetic gene synA21 was based on the amino acid sequence of A21 mAb (Genbank Accession No. AY077781 for V L and AY077783 for V H) and constructed in V L - (G4 S) 4 - V H mode.
A polyhistidine sequence (his6-tag) was introduced to c-terminus of the synthetic gene for facilitating antibody detection and purification. The tables of P. pastoris preferred codons were according to the results reported by several groups [18-20].
Synthesis of the synA21 gene
The synA21 gene was synthesized by recursive PCR (rPCR) strategy [21]. A total of 17 oligonucleotides were designed to have lengths between 55-83 nucleotides and overlaps of 19-23 base pairs with theoretical melting temperature of 55-60°C using the Oligo 6.0 software. The sequences encoding the V L-linker and V H-his6 were synthesized by first PCR with oligonucleotides P1-P8 and P9-P16 respectively. Amplification was performed with 10 nM of each internal oligonucleotides and 1 uM of each 5’-flanking oligonucleotides under optimal conditions: 95°C for 2 min; 30 cycles of 95°C, 56°C and 72°C each for 1 min; 72°C for 10 min. The two products were gel-purified, and equal molar of them were mixed for 10 cycles of amplification of 95°C, 56°C and 72°C each for 1 min. Then, the flanking oligonucleotides P0 and P16 were added for additional 20 cycles of amplification to generate the full-length gene.
Construction of expression vectors
The synA21 gene was cloned into the XhoI-EcoRI sites of the pPIC9 vector to generate the pPIC9/synA21 construct. The 1100bp BamHI-EcoRI digestion products containing the α-factor sequence and synA21 gene were cloned into the BamHI-EcoRI sites of the pPIC9K vector to generate the pPIC9K/synA21 construct. Similarly, the native scFv gene (scA21) was fused with a c-terminal his6-tag by PCR-amplification from the pCANTAB5E/scFv vector [8] and cloned into the pPIC9K vector to generate the pPIC9K/scA21 construct.
Transformation and screening for multicopy transformants
5-10 ug plasmid DNA was linearized with SalI and electrotransformed into P. pastoris strain GS115 (1.5 KV, 200 ohm, 25 uF, Biorad Gene Pulser). His+ transformants were selected on histidine-deficient MD plates. In vivo screening of multiple inserts was according to the Invitrogen pPIC9K Expression Manual and antibiotic G418 was used in three concentrations: 0.5, 1.5 and 3.0 mg/ml. The Mut+transformants were screened out by replica-plating the G418-resistant transformants on MD/MM plates. For small-scale culture experiments, single clone was cultured in 2 ml BMGY medium at 30°C overnight with shaking at 250 rpm. The cells were then resuspended in 2 ml BMMY medium to OD 600nm 1.0 to induce expression by addition of 1% methanol every 24h.
SDS-PAGE
200 ul culture supernatant was mixed with 1/9 volume of 100% TCA and incubated on ice for 30 min. After centrifugation (15000 g, 15 min, 4°C), the pellet was washed once with ice-cold acetone, air-dried, resuspended in 20 ul non-reducing loading buffer, and separated on 12% polyacrylamide gels. The gels were stained with coommssie brilliant blue R250 and the band densities were
evaluated by densitometric scanning. The scFv was quantified by comparing the signal densities present in lanes with that present in lanes containing known quantity of purified scFv. Quantitative ELISA
Supernatant samples were diluted 100-fold with 50 mM sodium carbonate buffer (pH 9.6) and coated on 96-well microtiter plate (Nunc) overnight at 4°C. To construct a standard reference curve, a series of dilutions containing 0–10 ng purified scFv were included in each assay. The plate was blocked with 1% non-fat milk in TPBS (PBS with 0.05% Tween 20) for 1h at room temperature. The plate was then incubated with mouse anti-histag antibody (1:2000, Qiagen) followed by HRP-conjugated goat-anti-mouse IgG (1:1000, Pierce) each for 1h at room temperature. The color reaction was developed by addition of the substrate solution (1 mg/ml o-phenylenediamine and 0.1% H2O2 in 0.1 M citrate buffer, pH 5.5) and stopped by 2M H2SO4. The absorbance at 490nm was measured in ELX800 Microaplate Reader (Bio-Tek Instruments Inc.). It was observed that there was a close agreement between the values obtained by SDS-PAGE and quantitative ELISA. Southern and Northern blotting analysis
The DNA fragment containing the 5’ untranslated mRNA leader sequence of AOX1 gene and the α-factor signal sequence was PCR-amplified from the pPIC9K vector with 5’-primer: ACA GAA GGA AGC TGC CCT GTC T and 3’-primer: AGC TTC AGC CTC TCT TTT CTC. This fragment was labeled with α-32P-dCTP using the Random Primer Labeling Kit (Takara) and used as the hybridization probe. Genomic DNA or total RNA was extracted from the P. pastoris transformants after 24h methanol induction using the Yeast Genomic DNA or Total RNA Isolation Kit (Watson’s Biotech). Southern blotting was according the procedures described by Hohenblum et al. [22] with some modifications. Both pPIC9K control DNA and genomic DNA were digested with BglII and XhoI, electrophoresed in 0.8% agarose, transferred to Hibond-N+ nylon membrane, and hybridized with 10ng labeled probes overnight at 65°C. For northern blotting, RNA samples (20ug each) were electrophoresed in 1.2% MOPS/formaldehyde agarose and the hybridization was preformed at 42°C. The blots were visualized by autoradiography and the band intensities were quantified by denstiometric scanning.
Conditions for scFv expression in shake flask culture
A single clone was cultured in 50 ml BMGY medium at 30°C overnight. To induce expression, cell aliquots were resuspended in 10 ml BMMY medium to OD600 nm 1.0 in 100 ml baffled flasks. The phosphate buffer was adjusted to pH 5.0-8.0 with 0.5 pH intervals. The induction was maintained for 6 days by addition of 1% methanol every 24h. If required, 2% casamino acids or 2.5 mM EDTA was added to the BMMY medium. At the desired time points, 0.2 ml cell aliquots were withdrawn and then replaced with equal amount of fresh medium.
Large-scale expression and purification of scFv
The best clone was cultured in 200ml BMGY at 30°C for 2 days. The cells were resuspended in 1L BMMY medium at pH 7.0 with 2% casamino acids and induced for 3 days. The supernatant was harvested by centrifugation (10,000 g, 30 min, 4°C), filtered through 0.22 um filter, dialyzed against binding buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.2), and loaded onto 3 ml Ni2+ Chelating Sephorase Fast Flow resins (Pharmacia Biotech) at a flow rate of 3 ml/min. The contaminant materials were washed away with binding buffer containing 0.01 M imidazole. Recombinant scFv was eluted with binding buffer containing 0.1M imidazole. The appropriate fractions were pooled, buffer exchanged to PBS and condensed by ultrafiltration using 10KDa cut-off membrane (Amicon, Millipore). Protein concentrations were determined by the Lowry
Protein Assay Kit after DOC and TCA precipitation using BSA as standard (Sigma). Determination of scFv binding affinity by ELISA
For direct binding assay, microtiter plate was coated with purified ErbB2 at 0.5, 0.25, 0.125, 0.63 ug/ml respectively. Serially 3-fold diluted scFv from 2.5*10-4 to 15 ug/ml concentration was added for 1h incubation. The bound scFv was detected with mouse anti-histag antibody and then with HRP-conjugated goat anti-mouse IgG as described before. The affinity constant was calculated using the formula K aff (M-1) = 1/2(2[Ab’]t - [Ab]t) [23]. For competitive ELISA, microtiter plate was coated with 0.1 ug/ml ErbB2. The mixture of A21 at 1.5 ug/ml and scFv at various concentrations were added for 1h incubation. The residual binding of A21 to ErbB2 was detected with HRP-conjugated goat anti-mouse IgG.
Internalization of scFv in SKBR3 Cells
SKBR3 cells were grown on coverslips in 6-well plates to 50% confluency and treated with 20 μg/ml scFv or 10 ug/ml A21 mAb at 4°C or 37°C each for 2 h. The cells were washed with ice-cold PBS and then with stripping buffer (500 mM NaCl, 0.1 M glycine, pH 2.5) if necessary, fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 for 10 min. The cells were saturated with 1% TPBS/BSA for 1h and then incubated with mouse anti-histag antibody (1:50) followed by FITC-conjugated goat anti-mouse IgG (1:200). The location antibody in cell membrane and cytoplasm was detected by fluorescence microscope with 400* magnification scale (Olympus).
Antibody internalization assay
The efficiency of antibody internalization was quantified according to the method described by Neve et al. [6]. Antibodies were labeled with I125 by the Chloramine-T method. 5*105 SKBR3 cells were seeded in 6-well plates and incubated with 2 ug radioiodinated antibodies (1.3-1.6 uCi/ug) at 37°C for 2h. The cells were rinsed 4 times with Hanks’ balanced salt solution, and 2 times with ice-cold stripping buffer. The cells were then lysed with 2 ml of 100 mM TEA for 4 min at 4°C. Radioactivities of the rinses and the acid-washed cell lysate were determined.
RESULTS
Design of the synA21 gene
As we suspected that the poor codon bias in the native A21 scFv gene may be a possible cause of the inefficient translation and scFv production in P. pastoris, we designed a codon optimized gene to overcome this problem. Several groups have proposed the tables of P. pastoris preferred codons on the basis of different calculation methods [18-20]. Analysis of their results revealed that a number of amino acids have two preferred codons. Therefore, although the most-preferred codons were used during codon optimization in principle, the second-preferred codons were alternatively used to meet the following considerations: (1) elimination of stable secondary structures such as hairpins exceeding 6 base pairs especially in the vicinity of the 5’ and 3’ encoding regions of the corresponding mRNA sequence [27]; (2) prevention of probable depletion and congestion of tRNAs due to consecutively choosing the same codon for the frequently used amino acids; (3) remove of undesirable repeats and false priming events for facilitating subsequent gene synthesis. Finally, a total of 114 non-preferred codons in the native gene were replaced by the P. pastoris preferred codons in the synthetic gene, which represents 44% of the total amino acid sequence (Fig.1). As a result, codon optimization significantly reduced the overall G+C content of the full-length gene from 56% to 43%.
Construct of the synA21 gene
The synA21 gene was synthesized by rPCR strategy [21] using a series of overlapping oligonucleotides, which are also shown in Fig.1. To reduce the possibly introduced errors during PCR amplification, the designed gene was subdivided into two fragments: the V L-linker fragment (480bp) assembled with oligonucleotides P1-P8 and the V H-his6tag fragment (370bp) with oligonucleotides P9-P16. The two rPCR products were then assembled to the full-length synA21 gene (830bp) by overlap extension PCR (oePCR) with flanking primers P0 and P16. The synA21 gene was then cloned to the XhoI-EcoRI sites of the pPIC9K vector as in frame fusions with the α-factor signal sequence under the control of AOX promoter. Ten randomly picked clones were submitted to DNA sequencing analysis. Two of them had correct coding sequences and the others had at least one error, typically with singe point mutants or short deletions.
Effect of codon optimization on scFv expression
To test the effect of codon optimization on scFv expression, both the native and synthetic constructs were screened for multicopy clones by increasing the antibiotic G418 concentrations. The synthetic gene typically yielded 6-10 mg/L scFv in BMMY medium after methanol induction for 3 days (Fig.2). The mean expression levels of the transformants resistant to 0.5, 1.5 and 3.0 mg/ml G418 were 7.9, 8.5 and 8.4 mg/L respectively. Statistics analysis revealed that there was no significant difference in the scFv levels among three groups of the transformants. This yield was approximately 3-5 fold increase in comparison with the 1-2 mg/L scFv level of the native gene. The higher copy number in the more G418-resistant clones was further confirmed by southern blotting analysis. Typically, the clones resistant to 1.5 mg/ml G418 acquired 3-5 copies and the clones resistant to 3.0 mg/ml G418 acquired more than 5 copies, basically in accordance with the results proposed in the Pichia pPIC9K Expression Manual (Invitrogen). These data suggested that copy number had no obvious effect on the scFv expression.
To precisely evaluate the steady-state mRNA levels of the native and synthetic constructs, total RNA was extracted after 24 methanol induction and analyzed by northern blotting using the DNA probe containing the 0.4 Kb fragment of the 5’ untranslated AOX1 mRNA leader sequence and the α-factor signal sequence. The use of this probe ensured the cross-hybridization of the mRNAs of both constructs strictly with the same efficiency. Figure 3 shows that all of the transformants had the full-length transcripts of the expected size (1.4 Kb) without obvious degradation or premature polyadenylation. The mRNA levels of the synthetic construct were only 1.2-1.4 fold higher than that of the native construct. However, at the protein level, a significant improvement of approximately 5-fold was observed for the synthetic gene (data not shown). In addition, both constructs of two gene copies showed similar mRNA levels to that with one copy. Therefore, codon optimization did not appear to significantly influence the gene transcription.
Effect of culture pH and medium composition on scFv expression
The effect of culture pH on the cell growth and scFv expression was investigated by a time-course experiment in BMMY medium to reduce protease activities. The results showed a basically positive correlation between the cell growth and scFv accumulation. At all of the tested pH conditions from 5.0 to 8.0, the viable cell densities displayed a rapid increase during the first 72h induction and almost kept a platform of approximately 30-40 OD600 nm afterward until 144h induction. The scFv accumulation reached the maximal level during 72-120h induction and began to decline afterward. It was also observed that the best scFv production was achieved at pH 6.5-7.0 with 10-12 mg/L level, which was 1.2-1.3 fold higher than that at pH 6.0, although the cell growth rates were very
similar at these pH conditions. Extremely at pH above 8.0, the cell growth retained 70% level but the scFv production reduced to only 40% level.
The effect of casamino acids and EDTA on the scFv expression was shown in Fig.4. The addition of 2% casamino acids in BMMY medium increased the scFv yield approximately 1.5-fold. This improvement may partly result from the increased cell growth rate, as the cell density also increased 1.2-fold. However, the addition of 2.5 mM EDTA exhibited no effect on cell growth while the scFv production reduced to 80% level. The best clone produced up to 15 mg/L scFv after 96h methanol induction in BMMY medium at pH7.0 supplied with 2% casamino acids.
Purification of scFv
As P. pastoris cells only secreted small amounts of extracellular proteins into the culture supernatant, the secreted his6-tagged scFv was easily purified by one-step Ni2+ chelating chromatography. It was found that recombinant scFv bound to the resins with high efficiency after the dialyzed supernatant was loaded onto the column, because the scFv band almost disappeared in the flow-through fractions (Fig.5 lane 1 and 2). After the nonspecifically bound contaminants were washed away with binding buffer containing 0.01 mM imidazole (Fig.5 lane 4-8), the scFv was eluted with 0.1 M imidazole (Fig.5 lane 9-14). The purified scFv showed a homogeneous band with apparent molecular weight of approximately 30 KDa as examined by SDS-PAGE, corresponding to the predicted size of 29 KDa. The amount, purity and recovery yield of scFv for each purification step are summarized in Table 1. A typical purification process allowed for the purification of 14.1 mg scFv with the purity above 95% and the recovery yield of about 90% from 1L induced supernatant.
Binding affinity of scFv for ErbB2
The binding affinity of scFv for purified ErbB2 antigen was determined and compared with that of the parent A21 mAb by two ELISA methods. In the direct binding assay, the scFv reacted to ErbB2 in a concentration-dependent manner (Fig.6A). The affinity constant K aff was calculated to be 0.45*108, 1.16*108, 0.67*108 L/mol respectively. The average K aff was 0.76*108 L/mol and the dissociation constant K D was 13.1 nM. In the competitive ELISA, the binding of A21 for ErbB2 at 10 nM concentration was half-inhibited by the scFv at 61 nM concentration (Fig.6B). As the K D of A21 had been determined to be 1.8 nM [7], the K D of scFv was calculated to be 11.0 nM, slightly lower than the result of direct ELISA.
Internalization of scFv by SKBR3 cells
The purified scFv was tested for its ability to undergo receptor-mediated internalization after binding to the ErbB2-overexpressing SKBR3 cells. A strong membrane staining was observed in the cells treated with the scFv at 4°C as visualized by immunofluorescence microscope, indicating the scFv could specifically bind to the cell-surface ErbB2 receptor (Fig.7A). In contrast, an obviously intracellular staining was found after in the cells were treated with the scFv at 37°C for 2h,indicating the bound scFv could undergo efficient internalization (Fig.7B). Similarly, the A21 mAb could also be internalized efficiently into the cytoplasm after binding to the SKBR3 cells (Fig.7C). As a negative control, an anti-GPIb mAb showed no specific immunofluorescent staining (data not shown).
Further I125-labeled antibody internalization assay showed that the distribution of the scFv between the cell surface and intracellular compartment was slightly higher than that of A21 mAb after treatment at 37°C for 2h, with 46.6% and 43.4% being intracellular respectively (Fig.8). As a control, only 21.6 % of 4D5 mAb was internalized, in accordance to the results of humanized
4D5—Herceptin [6]. Therefore, the internalizing ability of A21 mAb and its scFv was approximately 2-fold higher than that of 4D5.
DISCUSSION:
In this study, we evaluated the utility of codon optimization to improve the expression of an anti-ErbB2 scFv in P. pastoris. We designed the full-length scFv gene by choosing the most or second preferred codons while avoiding the formation of stable secondary structures in the corresponding mRNA sequence. Resultingly, codon optimization moderately increased the scFv expression level 3-5 fold and up to 10 mg/L in the standard shaker flask cultures. Although this increase magnitude is somewhat lower than those of usually above 5-10 fold published for codon optimization studies in P. pastoris[20, 24, 25], it is still higher than the results of some other reports [26, 27].
The translational efficiency hypothesis related to translation initiation and elongation rates has been well accepted for explaining the codon usage bias in the prokaryotes and unicellular eukaryotes [28]. Northern blotting revealed that the mRNA levels were similar between the native and synthetic scFv constructs, suggesting that the increased expression effect was most attributable to the enhancement of post-transcriptional processing. As both the native and synthetic genes were placed after the α-factor secretion peptide and not in the vicinity of translation start codon context, we speculated that the increased scFv expression by codon optimization should be mainly due to the enhanced efficiency of translation elongation instead of translation initiation. This is experimentally supported by the findings that the amount of rare codons positively correlated with the decreased mRNA stability and translation elongation rate in the yeast S. cerevisiae[29, 30].
In addition to codon choice, mRNA structure can also influence the translation efficiency in yeasts. The specific secondary structures formed or lost near the mRNA untranslated region and the start codon were shown to significantly influence the mRNA degradation rate and translation initiation efficiency [31]. However, less data are available regarding the presence or absence of secondary structures in the coding region affecting the mRNA stability and translation. It was reported that optimization of the G+C content alone despite the actual codon bias could significantly improve the recombinant protein expression in P. pastoris as well as in mammalian cells [20, 32]. Actually, as P. pastoris prefers A/T–ended codons while mammals tend towards G/C-ended codons, codon optimization of a mammalian-derived gene into the P. pastoris preferred one usually leads to the relatively low G+C content accompanied with the mRNA of less stable secondary structures. Regardless of the mechanisms to be further studied, our data suggest that codon optimization and mRNA structure reduction can have positive effects on heterologous protein expression in P. pastoris.
Our attempt to improve the scFv expression by increasing the gene copies of the synthetic gene was found to be less effective. Woo et al. 2003 also reported that there was no difference in the expression level of a scFv-fused immunotoxin between the clones of single and double copies [25]. It seems that other rate-limiting factors including protein folding within the endoplasmic reticulum (ER) and secretion signal processing may also determine the scFv secretion ability. Recently, the unfolded protein response concerning a series of proteins was identified to prevent the secretion enhancement of heterologous proteins by increasing gene copies in the yeasts [22, 33]. Consistently, several groups have reported that co-expression of chaperone proteins such as BiP, PDI and SEC4 to assist folding and secreting remarkably improved the expression of recombinant proteins
including scFv fragments [34-36]. Further studies are needed to investigate whether this co-expressing strategy can help to express the A21 scFv in P. pastoris.
As cell density and protease activity may have impactful influence on the yield of secreted proteins in P. pastoris, we investigated the effect of culture pH and medium composition on scFv production. It was found that scFv accumulation positively correlated with the cell growth in BMMY medium and achieved the highest level at pH 6.5-7.0. In contrast, Shi et al. did not observe a correlation between the cell growth, protease activity and scFv accumulation, as the anti-serpin scFv was sensitive to extracellular proteolysis [14]. Although P. pastoris can secrete several types of extracellular proteases into culture medium, it seems that the inclusion of yeast extract and peptone provides efficient prevention of our scFv from protease degradations. This is supported by our observations that the addition of casamino acids only limitedly increased the scFv production and even the addition of EDTA led to a negative effect. These data thus provide useful parameters for optimizing fermentation conditions to achieve higher scFv production.
The ability of anti-ErbB2 antibodies to inhibit tumor cell growth differ much with their specifically recognized epitopes and associate commonly with their abilities to induce ErbB2 receptor endocytosis [37, 38]. Although the affinity of the A21 scFv expressed in P. pastoris was 6-fold lower than that of A21 mAb and approximately 100-fold lower than that of 4D5, the internalizing ability of the scFv was slightly higher than A21 mAb and 2-fold better than 4D5. Our date confirmed the findings that the internalizing efficiency of anti-ErbB2 antibodies did not necessarily depend on either affinity or bivalency [6]. The A21 scFv should be especially useful for construction of fusion molecules for delivery of drugs, toxins or DNA into the cytoplasm for ErbB2-based immunotherapy.
Acknowledgments
This work is supported by Hi-Tech Research and Development Program (“863” Program) of the Ministry of Science and Technology of China (No. 2001AA215381) and Specialized Research Fund for the Doctoral Program of Higher Education (No. 20020358048).
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Figure legends:
Fig.1 Design and construction of the synA21 gene. The synthetic gene is based on the primary amino acid sequence of the A21 scFv and flanked upstream by the α-factor signal sequence and downstream by the his6-tag sequence. The DNA sequences were aligned and the changed codons were labeled out. For gene synthesis by rPCR, seventeen overlapping oligonucleotides (P0-P16) were designed, which are shown in dotted lines for forward primers and solid lines for backward primers. Restriction enzyme sites introduced for vector construction are underlined.
Fig.2 Expression of scFv in P. pastoris. Transformants of the native or synthetic constructs resistant to different concentrations of G418 (0.5, 1.5, 3.0 mg/ml) were submitted to methanol induction for 3 days. The amount of secreted scFv in culture supernatant was quantified by quantitative ELISA using mouse anti-histag antibody. The number of clones (n) analyzed is indicated.
Fig.3 Northern blotting analysis. Total RNA was extracted from the P. pastoris transformants after 24h methanol induction. 20 ug RNA was used for each sample. P32-labeled DNA probe representing the AOX1 mRNA 5’ leader sequence and the α-factor signal sequence were used for hybridization. The relative amount of mRNA was quantified by densitometric scanning. Lane1: GS115 negative control; Lane 2: native gene, one copy; Lane 3: native gene, tow copies; Lane 4: synthetic gene, one copy; Lane 5: synthetic gene, two copies.
Fig.4 Effects of casamino acids and EDTA on scFv expression. Two clones were used for methanol induction for 3 days in BMMY or BMMY with 2% casamino acids (CA) or 2.5 mM EDTA. The scfv level was determined by quantitative ELISA. Data were calculated from three independent experiments.
Fig.5 SDS-PAGE analysis of scFv purification. The secreted scFv was purified from the induced culture supernatant by one-step Ni2+ chelating affinity chromatography after dialysis. Lane 1: 200 ul supernatant; Lane 2: 200 ul supernatant in the flow-through fractions; Lane 3: Protein weight marker; Lane 4-8: fractions eluted with 0.01 M imidazole. Lane 9-14: fractions eluted with 0.1 M imidazole.
Fig.6 Binding affinity of purified scFv. (A) Direct binding assay. Purified ErbB2 antigen was used in 0.5, 0.25, 0.125, 0.63 ug/ml. The scFv was 3-fold diluted from 2.5*10-4 to 15 ug/ml. (B) Competitive ELISA. Purified ErbB2 was used in 0.1 ug/ml concentration. A21 at 10 nM and scFv at 2-fold serial dilutions from 0 to 1.28 uM were competed for binding to ErbB2.
Fig.7 Internalization of scFv in SKBR3 cells. SKBR3 cells were incubated with scFv at 4°C (A), scFv at 37°C (B) or A21 at 37°C each for 2h. After washed with PBS and then with stripping buffer (37°C only), the cells were fixed and permeabilized. The cell-surface bound and internalized antibodies were detected with mouse anti-histag antibody and FITC-conjugated goat anti-mouse IgG by fluorescence microscope.
Fig.8 Antibody internalization assay. SKBR3 cells were incubated with I125-labeled antibodies (4D5 control, A21 mAb, scFv) at 37°C each for 2h. The proportion of internalized antibody was
calculated as the ratio of cell lysate radioactivity to the sum of radioactivities of cell lysate and acid rinses.
Figure 2
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毕赤酵母表达系统研究进展 作者：齐连权， 陈薇， 来大志， 于长明， 王海涛 作者单位：军事医学科学院微生物学流行病学研究所,北京,100071 刊名： 中国生物工程杂志 英文刊名：JOURNAL OF CHINESE BIOTECHNOLOGY 年，卷(期)：2002,22(6) 被引用次数：11次 参考文献(21条) 1.Trinh L;Noronha S B;Fannon M Recovery of mouse endostatin producedby Pichia pastoris using expanded bed adsorption[外文期刊] 2000(04) 2.查看详情 3.Barr KA;Hopkins S A;Sreekrishna K Protocol for efficient secretion of HSA developed from Pichia pastoris 1992 4.Cereghino J L;Cregg J M Heterologous protein expression in the methylotrophic yeast Pichia pastoris[外文期刊] 2000(1) 5.Kjeldsen T;Pettersson A F;Hach M Secretory expression and characterization of insulin in Pichia pastoris[外文期刊] 1999(29) 6.Bewley M C;Tam B M;Grewal J X ray crystallography and massspectroscopy reveal that the N lobe of human transferrin expressed in Pichia pastorisis folded correctly but is glycosylated on serine 32 [外文期刊] 1999(08) 7.Kalidas C;Joshi L;Batt C Characterization of glycosylated variantsof beta lactoglobulin expressed in Pichia pastoris[外文期刊] 2001(03) 8.Briand L;Perez V;Huet J C Optimization of the production ofa honeybee odorant binding protein by Pichia pastoris[外文期刊] 1999(03) 9.Rydberg E H;Sidhu G;Vo H C Cloning mutagenesis and structural analysis of human pancreatic alpha amylase expressed in Pichia pastoris[外文期刊] 1999(03) 10.Guo R T;Chou L J;Chen Y C Expression in Pichia pastoris andcharacterization by circular dichroism and NMR of rhodostomin[外文期刊] 2001(04) 11.Zani M;Brillard Bourdet M;Lazure C Purification and characterization of active recombinant rat kallikrein rK9[外文期刊] 2001(02) 12.ChirulovaV;Cregg J M;Meagher M M Recombinant protein production in an alcohol oxidase defective strain of Pichia pastoris in fed batch fermentations[外文期刊] 1997 13.Hasslacher M;Schall M;Hayn M High level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[外文期刊] 1997(1) 14.Takahashi K;Takai T;Yasuhara T Effects of site directed mutagenesis in the cysteine residues and the N glycosylation motif in recombinant Der f 1on secretion and protease activity[外文期刊] 2001(04) 15.Boado R J;Ji A;Pardridge W M Cloning and expression in Pichia pastoris of a genetically engineered single chain antibody against the rat transferrin receptor[外文期刊] 2000(06)
Pichia酵母表达系统使用心得 甲醇酵母表达系统有不少优点，其中以Invitrogen公司的Pichia酵母表达系统最为人熟知，并广泛应用于外源蛋白的表达。虽然说酵母表达操作简单表达量高，但是在实际操作中，并不是每个外源基因都能顺利得到高表达的。不少人在操作中会遇到这样那样的问题，收集了部分用户在使用EasySelect Pichia Expression System这个被誉为最简单的毕赤酵母表达的经典试剂盒过程中的心得体会。其中Xiang Yang是来自美国乔治城大学（Georgetown University）Lombardi癌症中心（Lombardi Cancer Center），部分用户来自国内。 甲醇酵母部分优点： 1.属于真核表达系统，具有一定的蛋白质翻译后加工，有利于真核蛋白的表达； 2.AOX强效启动子，外源基因产物表达量高，表达产物可以达到每升数克的水平； 3.酵母培养、转化、高密度发酵等操作接近原核生物，远较真核系 统简单，非常适合大规模工业化生产； 4.可以诱导表达，也可以分泌表达，便于产物纯化； 5.可以甲醇代替IPTG作为诱导物，部分甲醇酵母更可以用工业甲醇替代葡萄糖作为碳源，生产成本低。 产品性能：优点——使用简单，表达量高，His-tag便于纯化；缺点——酵母表达蛋白有时会出现蛋白切割问题。 巴斯德毕赤酵母（Pichia pastoris）是一种能高效表达重组蛋白的酵母品种，一方面由于其是属于真核生物，因此表达出来的蛋白可以进行糖基化修饰，另一方面毕赤酵母生长速度快，可以将表达的蛋白分泌到培养基中，方便蛋白纯化。 毕赤酵母表达载体pPICZ在多克隆位点（MCR）3'端带有his-tag和c-myc epitopes，这些tag有利于常规检测和纯化，而且在MCR5'端引入了alpha factor（α-factor）用以分泌表达，并且在表达后α-factor可以自动被切除。在进行克隆的时候，如果你选择的是EcoRI，那么只需在目标蛋白中增加两个氨基酸序列即可完成。另外pPICZ系列选用的是Zeocin抗生素作为筛选标记，而诱导表达的载体需要甲醇——甲醇比一般用于大肠杆菌表达诱导使用的IPTG便宜。 第一步——构建载体 Xiang Yang：pPICZ系列有许多克隆位点可供选择，同时也有三种读码框以便不用的用户需要。 红叶山庄：有关是选择pPIC9K还是pPICZ系列？pPIC9K属于穿梭质粒，也可以在原核表达，而pPICZ系列比较容易操作，大肠和毕赤酵母均用抗Zeocin筛选（PIC9K操作麻烦一点，大肠用amp抗性，而毕赤酵母先用His缺陷筛选阳性克隆，在利用G418筛选多拷贝），而且对于大小合适（30—50KD）的蛋白在产量上是pPIC9K无法比拟的。 leslie：要做毕赤酵母表达实验，首先当然就要了解这个可爱的酵母了（椭圆形，肥嘟嘟的，十分可爱），她和大肠杆菌长得有较大区别（大肠杆菌是杆状的），因此在培养的过程中要区别这两种菌体，除了气味，浓度，颜色以外，也可以取样到显微镜中观测。大家做毕赤表达的时候应该都遇过这种情况吧，表达过程中染菌（我们实验室曾经污染过各种颜色形状的细菌，那真是一段可怕的经历），如果在不知情的情况下继续做下去，那可以就是浪费大把的
实验五酵母RNA的提取与鉴定 一、实验目的 1.掌握稀碱法分离酵母RNA的原理与操作过程。 2.了解RNA的组分并掌握定性鉴定的具体方法。 二、实验原理 1.酵母核酸中RNA含量较多，DNA含量则少于2%。 2.RNA可溶于碱性溶液，当碱被中和后，可加乙醇使其沉淀，有此可得粗RNA制品。 3.用碱液提取的RNA有不同程度的降解 三、实验仪器 1.离心机 2.水浴锅 3.电炉 4.烧杯 5.量筒 四、实验试剂 1.干酵母粉 2.0.2%氢氧化钠溶液：2g氢氧化钠溶于蒸馏水并稀释至1000ml。 3.乙酸 4.95%乙醇 5.无水乙醚
6.10%硫酸 7.氨水 9.5%硝酸银溶液：5g硝酸银溶于蒸馏水并稀释至100ml，贮于棕色瓶中。 1.苔黑酚-三氯化铁溶液： 将100mg苔黑酚溶于100ml浓盐酸中，再加入100mgFeCl 3.6H2O。 临用时配制。 五、实验步骤 1.RNA的提取： （1）称取2g干酵母粉于100ml烧杯中，加入 0.2%氢氧化钠溶液10ml，沸水浴加热30分钟，经常搅拌（如沸水浴过程中溶液蒸干可再加5~10ml氢氧化钠）。然后加入乙酸数滴，使提取液呈酸性（pH 试纸检验），离心10-15分钟（4000r.p.m）。 （2）取上清液，加入2倍体积的95%乙醇，边加边搅，加毕，静止，待完全沉淀，过滤。 （3）滤渣先用95%乙醇洗2次，每次约5毫升，再用无水乙醚洗2次，每次也约5ml。 （4）洗涤时可小心地用玻璃棒搅动沉淀。乙醚滤干后，滤渣即为粗RNA，可鉴定。 2.鉴定： （1）取上述RNA约 0.5g，加10%硫酸5ml,加热至沸1—2分钟，将RNA水解。
实验七酵母菌细胞大小的测定 一、实验目的 1.了解测量微生物大小的原理； 2.学习并掌握接目测微尺的校正方法及微生物大小的测定方法，增强微生物细胞大小的感 性认识。 二、实验材料 1.菌种：啤酒酵母（Saccharomyces cerevisiae）菌悬液，枯草芽孢杆菌（Bacillus subtilis） 染色标本片。 2.仪器或其他用具：显微镜，接目测微尺，镜台测微尺，载玻片，盖玻片 三、实验原理 微生物细胞的大小是微生物基本的形态特征，也是分类鉴定的依据之一。微生物细胞个体较小，需要在显微镜下借助于特殊的测量工具—测微尺来测定其大小。测微尺包括镜台测微尺和接目测微尺。 镜台测微尺是一张中央部分刻有精确等分线的载玻片，专门用于校定接目镜测微尺每小格的相对长度。通常，刻度的总长是1mm，被等分为100格，每格0.01mm（即10μm）。镜台测微尺不直接用来测量细胞的大小。 接目测微尺是一块可以放入接目镜的圆形小玻片，其中央有精确的等分刻度，有等分为50小格和100小格的两种。在测量时将接目测微尺放在目镜的隔板上，即可来测量经显微镜放大后的细胞物象。也有专用的目镜，里面已经安放好了接目测微尺。 由于接目测微尺所测量的是经显微镜放大后的细胞物象，因此，在不同的显微镜或不同的目镜和物镜组合放大倍数不同，接目镜测微尺每一小格所代表的实际长度也不一样。所以，在用接目测微尺测量微生物大小之前，必须先用镜台测微尺校定接目镜测微尺，以确定该显微镜在特定放大倍数的目镜和物镜下，接目镜测微尺每一小格所代表的实际长度，然后根据微生物细胞相当于的接目镜测微尺格数，计算出微生物细胞的实际大小。 图7-1测微尺的安装 图7-2目镜测微尺图7-3用镜台测微尺校正接目测微尺
食品中霉菌和酵母分布、检测和鉴定 福建省疾病预防控制中心马群飞 1. 霉菌和酵母的基本特性 霉菌和酵母这种称谓仅是为了方便起见，将小型真菌有真正菌丝的称为霉菌，没有菌丝的称酵母，并没有分类学上的依据。相对于低等的细菌来说，霉菌和酵母生长缓慢，竞争能力较弱，故霉菌和酵母常在不利于细菌生长繁殖的环境中形成优势菌群。由于霉菌和酵母的细胞较大，新陈代谢能力强，故10２～10４个酵母即可引起一克食物的变质，而细菌则需要100倍于此数的细胞。 通常霉菌和酵母适合在高碳低氮有机物如植物性物质上生存。适合的pH 3～8，有些霉菌可以在pH2，酵母在pH 1.5时生活。水活度要求0.99～0.61，霉菌0.85时最适宜，某些嗜渗酵母和霉菌常引起糖果类食品的变质。一般的霉菌的生长温度为20～30℃，部分霉菌可以在不低于－7℃的温度下生长。酵母一般在0～45℃时生长。耐热能力较差，酵母细胞55～56℃几分钟就被杀死。少数霉菌的孢子（如丝衣霉）则可在90℃中耐受 几分钟。霉菌和酵母很多可以耐受防腐剂。如乳酸、醋酸、CO ２和SO ２ 等。有些酵母酯酶 活性高并能合成B族维生素。 2. 食品中酵母的常见类群 在食品中能分离出各种酵母菌，但它们很可能没有什么意义，因为其中多数来源于外界的偶然污染。仅有非常有限的几个酵母属可使经过加工并正常生产工艺包装的食品腐败。比如抗SO ２ 熏蒸的酵母，就是饮料、葡萄酒变质的常见因素。耐受保鲜剂的毕赤酵母，高度嗜氧，可以形成泡菜和酱油的表面膜。 当然，如果不按照标准的生产程序进行食品生产，那么许多外界污染的酵母都将在食品中大量繁殖，这种情况下，就谈不上优势菌群问题了，也不必进行酵母的分类鉴定。处理方法只有一个，恢复正常的生产程序。 2.1 乳制品：鲜奶易因细菌污染而腐败，酵母菌不是重要问题。当鲜奶被加工成奶油、乳酪、酸奶等制品后，由于细菌被抑制，酵母可相应地成为优势菌。它们使奶油、乳酪产生怪味和气体，使黄油产生有味物质，并可使酸奶和酸乳酪腐败。在实验室中，汉逊德巴利酵母、布提利假丝酵母、多孢丝孢酵母和红酵母均能导致固体和液体乳酪的腐败。 2.2 肉类与肉制品：通常情况下，这类制品适于细菌的生长，酵母不是引起变质的主要原因。某些德巴利酵母可使冷冻的猪、牛肉香肠和午餐肉表面出现令人不愉快的粘滑感觉。
毕赤酵母表达实验手册 大肠杆菌表达系统最突出的优点是工艺简单、产量高、生产成本低。然而，许多蛋白质在翻译后，需经过翻译后的修饰加工，如磷酸化、糖基化、酰胺化及蛋白酶水解等过程才能转化成活性形式。大肠杆菌缺少上述加工机制，不适合用于表达结构复杂的蛋白质。另外，蛋白质的活性还依赖于形成正确的二硫键并折叠成高级结构，在大肠杆菌中表达的蛋白质往往不能进行正确的折叠，是以包含体状态存在。包含体的形成虽然简化了产物的纯化，但不利于产物的活性，为了得到有活性的蛋白，就需要进行变性溶解及复性等操作，这一过程比较繁琐，同时增加了成本。 与大肠杆菌相比，酵母是低等真核生物，具有细胞生长快，易于培养，遗传操作简单等原核生物的特点，又具有真核生物时表达的蛋白质进行正确加工，修饰，合理的空间折叠等功能，非常有利于真核基因的表达，能有效克服大肠杆菌系统缺乏蛋白翻泽后加工、修饰的不足。因此酵母表达系统受到越来越多的重视和利用。 大肠杆菌是用得最多、研究最成熟的基因工程表达系统，当前已商业化的基因工程产品大多是通过大肠杆菌表达的，其主要优点是成本低、产量高、易于操作。但大肠杆菌是原核生物，不具有真核生物的基因表达调控机制和蛋白质的加工修饰能力，其产物往住形成没有活性的包涵体，需要经过变性、复性等处理，才能应用。近年来，以酵母作为工程菌表达外源蛋白日益引起重视，主更是因为酵母是单细胞真核生物，不但具有大肠杆菌易操作、繁殖快、易于工业化生产的特点，还具有真核生物表达系统基因表达调控和蛋白修饰功能，避免了产物活性低，包涵体变性、复性等等间题［1］。 与大肠杆菌相比，酵母是单细胞真核生物，具有比较完备的基因表达调控机制和对表达产物的加工修饰能力，人们对酿酒酵母（Saccharomyces.Cerevisiae）分子遗传学方面的认识最早，酿酒酵母也最先作为外源基因表达的酵母宿主．1981年
酵母菌的死活细胞鉴别与镜检计数 一、实验目的 1.掌握鉴别酵母菌细胞死活的染色方法。 2.了解血球计数板的构造，掌握利用它进行酵母菌计数的方法。 二、实验步骤 1.酵母菌血球计数板镜检计数 1)取一块盖玻片，加盖在血球计数板中央计数室上方。 2)用滴管吸取菌悬液滴于盖玻片的边缘，通过毛细作用渗入计数室，注意不能有 气泡产生，然后放置于载物台，静止5min，使细胞全部沉降到其表面。 3)高倍镜镜检，数对角线上5个中方格中细胞的总数，再计算出菌悬液浓度。为 了减少误差，应注意对样品适当稀释，以每个小方格中平均4~6个细胞为佳； 另外，也可对同一样品重复计数，取其平均值。 附:计数板的结构及测定原理 利用血球技术板镜检技术是一种常用的计数方法。血球计数板是一块比普通载玻片厚的特质玻片，其上有四条凹槽，构成三个平台，中间比较宽，其中央又被一短横槽隔成两半，每半边各有一个计数区，其上有9个大方格，只有中央的一个大方格为计数室供计数用。这一大方格的长宽各为1mm。加盖盖玻片后，载玻片与盖玻片之间的距离为0.1mm，因此计数室的容积为0.1mm3。 目前血球计数板常用的是25格×16格型，其计数室被分成25个中格，其中每个中格又分为16个小格，故计数室共有400 小格。 计数时，首先把适当浓度的菌悬液注入计数室，然后在显微镜下计数，一般数对角线上5个中方格（共80个小方格）中细胞总数再根据下式求得菌悬液的浓度 细胞个数=5000A×B 式中A---5个中方格中细胞总数 B---菌悬液的稀释倍数 三、实验结果
个数:30 37 11 25 21 24 17 27 28 25
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毕赤酵母多拷贝表达载体试剂盒 用于在含多拷贝基因的毕赤酵母菌中表达并分离重组蛋白 综述： 基本特征： 作为真核生物，毕赤酵母具有高等真核表达系统的许多优点：如蛋白加工、折叠、翻译后修饰等。不仅如此，操作时与E.coli及酿酒酵母同样简单。它比杆状病毒或哺乳动物组织培养等其它真核表达系统更快捷、简单、廉价，且表达水平更高。同为酵母，毕赤酵母具有与酿酒酵母相似的分子及遗传操作优点，且它的外源蛋白表达水平是后者的十倍以至百倍。这些使得毕赤酵母成为非常有用的蛋白表达系统。 与酿酒酵母相似技术： 许多技术可以通用： 互补转化基因置换基因破坏另外，在酿酒酵母中应用的术语也可用于毕赤酵母。例如：HIS4基因都编码组氨酸脱氢酶；两者中基因产物有交叉互补；酿酒酵母中的一些野生型基因与毕赤酵母中的突变基因相互补，如HIS4、LEU2、ARG4、TR11、URA3等基因在毕赤酵母中都有各自相互补的突变基因。 毕赤酵母是甲醇营养型酵母： 毕赤酵母是甲醇营养型酵母，可利用甲醇作为其唯一碳源。甲醇代谢的第一步是：醇氧化酶利用氧分子将甲醇氧化为甲醛，还有过氧化氢。为避免过氧化氢的毒性，甲醛代谢主要在一个特殊的细胞器－过氧化物酶体－里进行，使得有毒的副产物远离细胞其余组分。由于醇氧化酶与O2的结合率较低，因而毕赤酵母代偿性地产生大量的酶。而调控产生醇过氧化物酶的启动子也正是驱动外源基因在毕赤酵母中表达的启动子。 两种醇氧化酶蛋白： 毕赤酵母中有两个基因编码醇氧化酶－AOX1及AOX2。细胞中大多数的醇氧化酶是AOX1基因产物。甲醇可紧密调节、诱导AOX1基因的高水平表达，较典型的是占可溶性蛋白的30%以上。AOX1基因已被分离，含AOX1启动子的质粒可用来促进编码外源蛋白的目的基因的表达。AOX2基因与AOX1基因有97%的同源性，但在甲醇中带AOX2基因的菌株比带AOX1基因菌株慢得多，通过这种甲醇利用缓慢表型可分离Muts菌株。 表达： AOX1基因的表达在转录水平受调控。在甲醇中生长的细胞大约有5%的polyA+ RNA 来自AOX1基因。AOX1基因调控分两步：抑制/去抑制机制加诱导机制。简单来说，在含葡萄糖的培养基中，即使加入诱导物甲醇转录仍受抑制。为此，用甲醇进行优化诱导时，推荐在甘油培养基中培养。注意即使在甘油中生长（去抑制）时，仍不足以使AOX1基因达到最低水平的表达，诱导物甲醇是AOX1基因可辨表达水平所必需的。 AOX1突变表型： 缺失AOX1基因，会丧失大部分的醇氧化酶活性，产生一种表型为Muts的突变株（methanol utilization slow），过去称为Mut，而Muts可更精确地描述突变子的表型。结果细胞代谢甲醇的能力下降，因而在甲醇培养基中生长缓慢。Mut+（methanol utilization plus）指利用甲醇为唯一碳源的野生型菌株。这两种表型用来检测外源基因在毕赤酵母转化子中的整合方式。 蛋白胞内及分泌表达： 外源蛋白可在毕赤酵母胞内表达或分泌至胞外。分泌表达需要蛋白上的信号肽序列，将外源蛋白靶向分泌通路。几种不同的分泌信号序列已被成功应用，包括几种外源蛋白本身分 制作者：陈苗商汉桥
实验二酵母菌的死活鉴定和显微镜计数 一、实验目的 学习血球计数板使用的原理与方法，学习区别死活酵母的方法。 二、原理 测定微生物数量方法很多，通常采用的有显微直接计数和平板计数法。 镜检计数法适用于各种单细胞菌体的纯培养悬浮液，菌体较大的酵母或霉菌孢子可采用血球计数板，一般细菌采用彼得罗夫?霍泽细菌计数板，两种计数板的原理和部件相同，只是细菌计数板较薄，可以使用油镜观察，而血球计数板较厚，不能使用油镜，故细菌不易看清。 血球计数板的构造 血球计数板是一块特制厚玻片。玻片上由四道槽构成三个平台，中间的平台分成两半，其上各刻一个相同而有一定面积的小方格网。方格的刻度有两种规格。一种是25×16，称为希里格式血球计数板，分为25大格，每大格又分为16小格；另一种是16×25，称为麦氏血球计数板，分16大格，每大格分为25小格。总数都是400小格（如图所示）。 每个大方格边长为1mm，则每一大方格的面积为1mm2，每个小方格的面积为1/400mm2，盖上盖玻片后，盖玻片与计数室底部之间的高度为0.1mm，所以每个计数室（大方格）的体积为0.1mm3，每个小方格的体积为1/4000mm3，使用血球计数板直接计数时，先要测定每个小方格（或中方格）中微生物的数量，再换算成每毫升（或每克样品）中微生物细菌的数量。
三、实验材料 1、菌种：酵母菌 2、仪器：显微镜、血球计数板 3、材料：盖玻片、吸水纸、滴管 4、染料：美蓝染液 四、实验步骤和内容 1、视待测菌液浓度，加无菌水做适当稀释，以每小格菌数5-10个为宜，准确计算稀释 倍数。 2、取清洁干燥的血球计数板，加盖玻片盖住网格和两边的槽。 3、将待测菌液充分摇匀后，用无菌吸管吸少许，由盖玻片边缘或槽内加入计数板来回推压盖玻片，使其紧贴在计数板上，计数室内不能有气泡。静置5-10分钟。 4、在低倍镜下找到小方格网后更换高倍镜观察计数，上下调动细螺旋，以便看到小室内不同深度的菌体。位于分格线上的菌体，只数两条边上的，其余两边不计数。如数上线就不数下线，数左边线就不数右边线。由于菌体在计数室中处于不同的空间位置，要在不同的焦距下才能看到，因为观察时必须不断调节微调螺旋，方能数到全部菌体，防止遗漏。凡酵母菌芽体达到母细胞一半时，即可作为两个菌体计算。 5、计数时若使用刻度为25×16（大格）的计数板，则数四角的4个大格（即100小格）内的菌数。如用刻度为16×25（大格）的计数板，除数四角的4个大格外，还需数中央1个大格的菌数（即80小格）。 6、每个样品重复计数2-3次，取其平均值，按下式计算样品中的菌数。 刻度为25×16（大格）的计数板：
山东大学实验报告2009年11月日 姓名郑冲冲系年级08级生科一班组别同组者张健康于政达 学号200800140207题目酵母菌的培养、形态观察、死活鉴定和子囊孢子的观察 一、目的要求 1、掌握酵母培养基的配置和酵母菌的接种方法 2、观察酵母菌的细胞形态及出芽生殖方式。 3、学习掌握区分酵母菌死、活细胞的实验方法。 4、学习并掌握酵母菌子囊孢子的观察方法。 二、基本原理 1、培养基：酵母菌的能源主要是糖，一般用麦芽汁琼脂培养基培养。但实验要求不是很严格时，可以用由酵母膏、蛋白胨、葡萄糖配置的培养基来培养；但酵母菌子囊孢子的观察要用麦氏培养基，其有利于子囊孢子的形成。 2、酵母菌的形态：酵母菌是多形的、不运动的单细胞真核微生物，菌体比细菌大。繁殖方式也较复杂，无性繁殖主要是出芽生殖；有性繁殖是通过接合产生子囊孢子。本实验通过用美蓝染色浸片和水-碘液来观察生活的酵母形态和出芽生殖方式。 3、美蓝染色液：美蓝为无毒性染料，其氧化型为蓝色，而还原型为无色。用它对酵母菌染色时，由于活细胞的新陈代谢作用，使细胞内具有较强的还原能力，能使美蓝从蓝色的氧化型变为无色的还原型，所以酵母的活细胞无色；对于死细胞或代谢缓慢的老细胞，则因它们无此还原能力或还原能力极弱，而被美蓝染成蓝色或淡蓝色。因此，用美蓝水浸片不仅可观察酵母的形态，还可以区分死、活细胞。 4、水-碘液染色液：该染液将革兰氏染液用碘液用水稀释4倍后得到的，亦可用于酵母形态和出芽生殖的观察。 4、子囊孢子的观察：酵母菌形成子囊孢子需要一定的条件，麦氏培养基有利于酿酒酵母子囊孢子的形成，能否形成子囊孢子及其形态是酵母菌分类鉴定的重要依据之一。 三、实验器材 1.菌种：酿酒酵母培养数天的酵母-麦氏培养基斜面 2.溶液与试剂：0.1% 吕氏碱性美蓝染液，5%孔雀绿，0.5%沙黄、95%乙醇，水-碘染液 3.培养基：酵母液体培养基，麦氏培养基。 4.仪器或其他用具：显微镜，载玻片，盖玻片，酒精灯、接种环等。 四、实验步骤 1、酵母培养基的配置：蛋白质2%，酵母膏1%葡萄糖2%，加冷水100Ml，自然PH，在115℃下高压蒸汽灭菌30min。 2、接种和培养：无菌操作下，在灭菌的培养基中倒入少许酵母菌液即可。在28℃下培养48h 左右。 3、美蓝染色操作： 1>在载玻片中央加一滴吕氏碱性美蓝染色液，用接种环挑取少量酵母菌液，混合均匀； 2>用镊子取一块盖玻片，先将一边与菌液接触，然后慢慢将盖玻片放下使其盖在菌液上，避免产生气泡； 3>将制片放置约3min，先低倍镜后高倍镜观察酵母形态和出芽情况，并根据颜色区别死活细胞。 4>染色开始到过30min期间，观察死活细胞数量的变化； 4、水-碘液浸片法：滴加一滴水-碘液在载玻片的中央，无菌操作挑取少许酵母菌液至于染
CHO细胞表达系统与酵母细胞表达系统比较 CHO细胞表达系统与毕赤酵母表达系统是当前发展前景看好的两个表达系统，为了能够更加直观地对两个表达系统有一定的认识，特意在此篇中对两个表达系统作一定的比较，从而能够更进一步的对两个表达系统有更深的了解 1．CHO细胞表达系统 （1）优点 CHO细胞属于成纤维细胞，既可以贴壁生长。也可以悬浮生长。目前常用的CHO细胞包括原始CHO和二氢叶酸还原酶双倍体基因缺失型(DHFR-)突变株CHO。近年来，为降低生产成本和减少血制品带来的潜在危害性，动物细胞生产开始使用无血清培养基(SFM)，但SFM往往导致细胞活力差，贴壁性差，分泌外源蛋白的能力差等缺点。另有研究者尝试将类胰岛素生长因子IGF基因和转铁蛋白基因转入CHO细胞获得能自身分泌必需蛋白的“超级CHO”，无需在培养基中转铁蛋白和胰岛素，细胞可在SFM 中生长良好。与其他表达系统相比，CHO表达系统具有以下的优点： (1)具有准确的转录后修饰功能，表达的蛋白在分子结构、理化特性和生物学功能方面最接近于天然蛋白分子； (2)既可贴壁生长，又可以悬浮培养，且有较高的耐受剪切力和渗透压能力； (3)具有重组基因的高效扩增和表达能力，外源蛋白的整合稳定； (4)具有产物胞外分泌功能，并且很少分泌自身的内源蛋白，便于下游产物分离纯化； (5)能以悬浮培养方式或在无血清培养基中达到高密度培养。且培养体积能达到1000L以上，可以大规模生产。 （2）存在的问题 在过去的几十年里，人类对动物细胞的培养技术进行了大量的研究开发，取得了很大进展，但是利用CHO细胞表达外源基因的技术水平尚不能满足生物药品的开发和生产的要求，目前上游工作中主要存在以下问题： ①构建的重组CHO细胞生产效率低，产物浓度亦低； ②某些糖基化表达产物不稳定，不易纯化； ③重组CHO细胞上游构建与下游分离纯化脱节，主要表现在上游构建时着重考虑它的高效表达，而对高教表达的产物是否能有效地提取出来，即分离纯化过程考虑较少； ④重组细胞培养费用昂贵，自动化水平低下。 2．毕赤酵母细胞表达系统 （1）特点 自1987年Gregg等首次在毕赤酵母中表达乙型肝炎表面抗原(HBsAg)到1995年，已有四十多种外源蛋白在毕赤酵母宿主菌中获得表达。而最近几年每年报道的在毕赤酵母中表达的外源基因就有几十种，且一年比一年多，与其它表达系统相比，毕赤酵母表达系统具有以下优势： 1）含有特有的强有力的AOX（醇氧化酶基因）启动子，用甲醇可严格地调控外源基因的表达； 2）表达水平高，即可在胞内表达，又可分泌型表达。毕赤酵母中，报道的最高表达量为破伤风毒素C为12g/l，一般大于1g/l。绝大多数外源基因比在细菌、酿酒酵母、动物细胞中表达水平高。一般毕赤酵母中外源基因都带有指导分泌的信号肽序列，使表达的外源目的蛋白分泌到发酵液中，有利于分离纯化； 3）发酵工艺成熟，易放大。已经有大规模工业化高密度生产的发酵工艺，且细胞干重达100g/l 以上，表达重组蛋白时，已成功放大到10000升； 4）培养成本低，产物易分离。毕赤酵母所用发酵培养基十分廉价，一般碳源为甘油或葡萄糖及甲
毕赤酵母多拷贝表达载体试剂盒 用于在含多拷贝基因的毕赤酵母菌中表达并分离重组蛋白 综述： 基本特征： 作为真核生物，毕赤酵母具有高等真核表达系统的许多优点：如蛋白加工、折叠、翻译后修饰等。不仅如此，操作时与E.coli及酿酒酵母同样简单。它比杆状病毒或哺乳动物组织培养等其它真核表达系统更快捷、简单、廉价，且表达水平更高。同为酵母，毕赤酵母具有与酿酒酵母相似的分子及遗传操作优点，且它的外源蛋白表达水平是后者的十倍以至百倍。这些使得毕赤酵母成为非常有用的蛋白表达系统。 与酿酒酵母相似技术： 许多技术可以通用： 互补转化基因置换基因破坏另外，在酿酒酵母中应用的术语也可用于毕赤酵母。例如：HIS4基因都编码组氨酸脱氢酶；两者中基因产物有交叉互补；酿酒酵母中的一些野生型基因与毕赤酵母中的突变基因相互补，如HIS4、LEU2、ARG4、TR11、URA3等基因在毕赤酵母中都有各自相互补的突变基因。 毕赤酵母是甲醇营养型酵母： 毕赤酵母是甲醇营养型酵母，可利用甲醇作为其唯一碳源。甲醇代谢的第一步是：醇氧化酶利用氧分子将甲醇氧化为甲醛，还有过氧化氢。为避免过氧化氢的毒性，甲醛代谢主要在一个特殊的细胞器－过氧化物酶体－里进行，使得有毒的副产物远离细胞其余组分。由于醇氧化酶与O2的结合率较低，因而毕赤酵母代偿性地产生大量的酶。而调控产生醇过氧化物酶的启动子也正是驱动外源基因在毕赤酵母中表达的启动子。 两种醇氧化酶蛋白： 毕赤酵母中有两个基因编码醇氧化酶－AOX1及AOX2。细胞中大多数的醇氧化酶是AOX1基因产物。甲醇可紧密调节、诱导AOX1基因的高水平表达，较典型的是占可溶性蛋白的30%以上。AOX1基因已被分离，含AOX1启动子的质粒可用来促进编码外源蛋白的目的基因的表达。AOX2基因与AOX1基因有97%的同源性，但在甲醇中带AOX2基因的菌株比带AOX1基因菌株慢得多，通过这种甲醇利用缓慢表型可分离Muts菌株。 表达： AOX1基因的表达在转录水平受调控。在甲醇中生长的细胞大约有5%的polyA+ RNA 来自AOX1基因。AOX1基因调控分两步：抑制/去抑制机制加诱导机制。简单来说，在含葡萄糖的培养基中，即使加入诱导物甲醇转录仍受抑制。为此，用甲醇进行优化诱导时，推荐在甘油培养基中培养。注意即使在甘油中生长（去抑制）时，仍不足以使AOX1基因达到最低水平的表达，诱导物甲醇是AOX1基因可辨表达水平所必需的。 AOX1突变表型： 缺失AOX1基因，会丧失大部分的醇氧化酶活性，产生一种表型为Muts的突变株（methanol utilization slow），过去称为Mut，而Muts可更精确地描述突变子的表型。结果细胞代谢甲醇的能力下降，因而在甲醇培养基中生长缓慢。Mut+（methanol utilization plus）指利用甲醇为唯一碳源的野生型菌株。这两种表型用来检测外源基因在毕赤酵母转化子中的整合方式。 蛋白胞内及分泌表达： 外源蛋白可在毕赤酵母胞内表达或分泌至胞外。分泌表达需要蛋白上的信号肽序列，将外源蛋白靶向分泌通路。几种不同的分泌信号序列已被成功应用，包括几种外源蛋白本身分
作者：非成败 作品编号：92032155GZ5702241547853215475102 时间：2020.12.13 毕赤酵母表达系统 Mut+和Muts 毕赤酵母中有两个基因编码醇氧化酶——AOX1及AOX2，细胞中大多数的醇氧化酶是AOX1基因产物，甲醇可紧密调节、诱导AOX1基因的高水平表达，较典型的是占可溶性蛋白的30%以上。AOX1基因调控分两步：抑制/去抑制机制加诱导机制。简单来说，在含葡萄糖的培养基中，即使加入诱导物甲醇转录仍受抑制。为此，用甲醇进行优化诱导时，推荐在甘油培养基中培养。注意即使在甘油中生长(去抑制)时，仍不足以使AOX1基因达到最低水平的表达，诱导物甲醇是AOX1基因可辨表达水平所必需的。AOX1基因已被分离，含AOX1启动子的质粒可用来促进编码外源蛋白的目的基因的表达。AOX2基因与AOX1基因有97%的同源性，但在甲醇中带AOX2基因的菌株比带AOX1基因菌株慢得多，通过这种甲醇利用缓慢表型可分离Muts菌株。在YPD(酵母膏、蛋白胨、葡萄糖)培养基中，不论是Mut+还是Muts其在对数期增殖一倍的时间大约为2h。Mut+和Muts菌株在没有甲醇存在的情况下生长速率是一样的，存在甲醇的情况下，Mut+在对数期增殖一倍的时间大约为4至6个小时，Muts在对数期增殖一倍的时间大约为18个小时。 菌株GS115、X-33、KM71和SMD1168的区别 GS115、KM71和SMD1168等是用于表达外源蛋白的毕赤酵母受体菌，与酿酒酵母相比，毕赤酵母不会使蛋白过糖基化，糖基化后有利于蛋白的溶解或形成正确的折叠结构。GS115、KM71、SMD1168在组氨酸脱氢酶位点(His4)有突变，是组氨酸缺陷型，如果表达载体上携带有组氨酸基因，可补偿宿主菌的组氨酸缺陷，因此可以在不含组氨酸的培养基上筛选转化子。这些受体菌自发突变为组氨酸野生型的概率一般低于10-8。GS115表型为Mut+，重组表达载体转化GS115后，长出的转化子可能是Mut+，也可能是Muts(载体取代AXO1基因)，可以在MM和MD培养基上鉴定表型。SMD1168和GS115类似，但SMD1168基因组中的Pep4基因发生突变，是蛋白酶缺陷型，可降低蛋白酶对外源蛋白的降解作用。 其中X-33由于是野生型，因此耐受性比较好，如果担心转化率的话可以考虑这种酵母菌，而X33与GS115一样都是属于MUT＋表现型，也就是说可以在含甲醇的培养基中快速生长，但是据说会对外源基因表达有影响， KM71的亲本菌在精氨酸琥珀酸裂解酶基因(arg4)有突变，在不含精氨酸的培养基中不能生长。用野生型ARG4基因(约2kb)插入到克隆的野生型AOX1基因的BamHI(AOX1基因15/16密码子)及SalI(AOX1基因227/228密码子)位点，取代了AOX1基因16-227密码子，此结构转化至KM71亲本菌(arg4his4)中，分离产生KM71 MutsArg+His-菌株，Arg+转化子遗传分析显示野生型AOX1被aox1::ARG4结构所取代，所以KM71所有转化子都是Muts 表型。AOX1位点没有被完全缺失，理论上可用你的目的结构通过基因取代方法替换
7.2 用酵母菌研究一个种群 实验原理 酵母菌繁殖快，是单细胞个体，常被用来研究种群。我们将观察在试管内肉汤培养基中的酵母菌种群的生长情况。 酵母菌的种群属于封闭种群类型。在自然条件下，开放种群的大小会随着生物个体的迁入或迁出而变大变小。在开放种群中，各种物质可通过种群进行循环。但在封闭种群中，情况有些不同，测定封闭种群的增长率比开放种群的增长率要容易得多。用浊度计测定培养液的浑浊度，就能知道酵母菌种群是如何随时间而发生变化的。通过细胞计数就可以知道酵母菌细胞的数量变化与浑浊度之间的关系。 目的要求 通过实验观察，说明种群是如何随时间而发生变化的。 学习酵母菌计数的方法以及取样法。 材料用具（2人一组） 2副护目镜；2支16mm×150mm有螺旋盖的试管，每支盛有10ml无菌肉汤培养液；2支18mm×150mm试管；盖玻片；有标尺的载玻片（2mm×2mm方格）；有刻度的吸量管（1ml）;滴管；比浊计或比色计；显微镜；试管架；玻璃标记笔；米尺；4张半对数坐标纸。 实验方法 请仔细阅读实验并提出3种假设，说明种群如何随时间而发生变化。把这些假设记在你的记录本上，并用你在实验中收集的数据对它们做出评价。 本实验采用的方法叫取样法—通过对样品中的酵母菌计数以估计试管中的种群大小。还可根据试管中培养液的浑浊度获得这一估算值。 实验步骤 实验从第0天—第7天。 （一）第0天： 1、在你的记录本上画好与表2.1类似的数据表。 2、用标记笔把两支螺旋盖的试管标上A和B，并在每支试管上标上你的组别。 表2.1酵母菌细胞的数目
3、教师将把0.1ml酵母菌贮存用培养物注入试管A中，轻轻倒转试管几次使酵母细胞分布均匀。试管B不加任何东西。将试管盖稍微拧松并将两支试管放在教师指定的地方。设置试管B的目的是什么？ 4、试管A中为刚开始增长的酵母菌新种群。就下周期间你认为可能会发生的变化评论你的假设。 5、为了确定酵母菌种群的增长速率，必须在实验过程中对酵母菌进行计数。拧紧试管盖将试管A轻轻倒转几次使酵母菌细胞分布均匀，然后用滴管从试管A中取出一滴培养液移到载玻片方格上，小心盖上盖玻片，不要有气泡。在显微镜高倍镜下进行镜检。 注意：观察酵母菌细胞时光线不要太强。 6、为了计算中央方格内酵母菌细胞的数目，先将方格左上部置于高倍镜下并记下酵母菌细胞数目，接着按顺时针方向分别统计方格右上部、右下部和左下部的酵母菌数，直到把整个方格内全部酵母菌数量统计完为止。要确保你所观察到的是酵母菌细胞而不是其他的什么东西。酵母菌细胞常常粘附在一起，但可以数出任何一个分离团块中的每一个细胞，酵母菌出芽时的芽体也应算为独立的个体。 7、至少要数300个细胞，如果少于300就应在原方格周围的另一方格内进行计数，直到达到300为止。用细胞数除以方格数即可得到每方格内的平均数。 8、为了知道每立方厘米（cm3）体积(1ml)内的细胞数，可用计得的细胞数乘以2500。这是因为每方格的面积是2mm×2mm,盖玻片下的培养液厚度是0.1mm,所以每方格的体积就是 2mm×2mm×0.1mm=0.4mm3,而1cm3=1000mm3,因此：细胞数 1000mm32500×细胞数 为了知道试管A中的细胞总数，可将最终获得的细胞总数乘以10，因为试管A中含有10ml 培养液。 9、让同组的另一人对一个新样品做同样的工作，将结果写在记录本上并计算两次计数的平均值。把第0天观察到的种群大小填写在你的数据表中。 10、对试管B重复步骤5—9。 11、使用比浊计测定两试管的浑浊度。算出读数的平均值，并将第0天的平均值写入你的资料表和班长的表格中。当酵母菌种群增长时你预测会出现什么情况？把你的预测写入记录本。 （二）第1—7天 13、多次倒转试管A使酵母菌细胞分布均匀，测定浑浊度，将第1天的平均值记在你的资料表和班长的表格中，对试管B重复同样的工作。 14、分别使用试管A和试管B中的一滴培养液重复步骤5—9的计数工作，并将第1天的结果写入你的资料表和班长的表格中。
毕赤酵母表达经验总结 甲醇酵母表达系统有不少优点，其中以Invitrogen公司的Pichia酵母表达系统最为人熟知，并广泛应用于外源蛋白的表达。虽然说酵母表达操作简单表达量高，但是在实际操作中，并不是每个外源基因都能顺利得到高表达的。不少人在操作中会遇到这样那样的问题，生物通编者特地收集了部分用户在使用EasySelect Pichia Expression System这个被誉为最简单的毕赤酵母表达的经典试剂盒过程中的心得体会。其中Xiang Yang是来自美国乔治城大学（Georgetown University）Lombardi癌症中心（Lombardi Cancer Center），部分用户来自国内。 甲基酵母部分优点与其他真核表达系统比较与原核表达系统比较 1.属于真核表达系统，具有一定的蛋白质翻译后加工，有利于真核蛋白的表达优点-+ 2.AOX强效启动子，外源基因产物表达量高，可以达到每升数克表达产物的水平++++ 3.酵母培养、转化、高密度发酵等操作接近原核生物，远较真核系统简单，非常适合大规模工业化生产。+++= 4.可以诱导表达，也可以分泌表达，便于产物纯化。=+ 5.可以甲醇代替IPTG作为诱导物，部分甲醇酵母更可以甲醇等工业产物替代葡萄糖作为碳源，生产成本低++++ + 表示优胜于；- 表示不如；= 表示差不多 EasySelect Pichia Expression System 产品性能： 优点——使用简单，表达量高，His-tag便于纯化 缺点——酵母表达蛋白有时会出现蛋白切割问题 全面产品报告及心得体会： 巴斯德毕赤酵母（Pichia pastoris）是一种能高效表达重组蛋白的酵母品种，一方面由于其是属于真核生物，因此表达出来的蛋白可以进行糖基化修饰，另一方面毕赤酵母生长速度快，可以将表达的蛋白分泌到培养基中，方便蛋白纯化。 毕赤酵母表达载体pPICZ在多克隆位点（MCR）3'端带有his-tag和c-myc epitopes，这些tag有利于常规检测和纯化，而且在MCR5'端引入了alpha factor（α-factor）用以增加表达，并且在表达后α-factor 可以自动被切除。在进行克隆的时候，如果你选择的是EcoRI，那么只需在目标蛋白中增加两个氨基酸序列即可完成。另外pPICZ系列选用的是Zeocin抗生素作为筛选标记，而诱导表达的载体需要甲醇——甲醇比一般用于大肠杆菌表达诱导使用的IPTG便宜。 第一步——构建载体 Xiang Yang：pPICZ系列有许多克隆位点可供选择，同时也有三种读码框以便不用的用户需要。 红叶山庄：有关是选择pPIC9K还是pPICZ系列？pPIC9K属于穿梭质粒，也可以在原核表达，而pPICZ系列比较容易操作，大肠和毕赤酵母均用 抗Zeocin筛选（PIC9K操作麻烦一点，大肠用amp抗性，而毕赤酵母先用His缺陷筛选阳性克隆，在利用G418筛选多拷贝），而且对于大小合适（30—50KD）的蛋白在产量上是pPIC9K无法比拟的。leslie：要做毕赤酵母表达实验，首先当然就要了解这个可爱的酵母了（椭圆形，肥嘟嘟的，十分可爱），她和大肠杆菌长得有较大区别（大肠杆菌是杆状的），因此在培养的过程中要区别这两种菌体，除了气味，浓度，颜色以外，也可以取样到显微镜中观测。大家做毕赤表达的时候应该都遇过这种情况吧，表达过程中染菌（我们实验室曾经污染过各种颜色形状的细菌，那真是一段可怕的经历），如果在不知情的情况下继续做下去，那可以就是浪费大把的时间了。 基本熟悉了毕赤酵母，了解了她生长的喜好（多糖偏酸环境），生长的周期等等情况后，当然更多的精力还是应该花在表达的目的蛋白上，我的表达蛋白有些恐怖，有100KD，本来当然应该放在大肠杆菌中表达，但是为了分泌表达（其实后来发现大肠杆菌pET系列分泌表达系列也不错）和糖基化修饰（主要是这个方面，因为我的蛋白是人源的，表达出来用于酵母双杂，因此需要有完备的糖基化修饰）。这样我的DNA片段由于较长，所以在做克隆的时候也要非常小心，需要注意的是： ①酶切位点不能出现在目的DNA片段中——如果片段长无法避免，可以采用平末端连接； ②虽然α-factor可以自动切除，但是在设计表达的时候，如果在N端不能出现任何多余的aa（比如药物蛋白表达），需要特别留意（说明书上有详细说明：P13）； ③有三种不同的读码框（对于pPICZα系列来说就是对上α-factor序列），在设计克隆的时候要反复确