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基于转铁蛋白受体(TfR1)的肿瘤与脑部疾病靶向治疗研究进展人转铁蛋白受体（TfR1)在不同组织器官中普遍表达，其主要功能是协助转铁蛋白在细胞和血脑屏障内外转运，维持细胞铁平衡。
在肿瘤细胞中以及血脑屏障中，TfR1的表达水平明显高于正常细胞组织，因此，TfR1被认为是肿瘤靶向治疗和脑部疾病靶向治疗的重要靶点。
基于TfR1靶向治疗的药物载体主要有转铁蛋白（Tf）、抗TfR1抗体、TfR1结合肽，这些生物大分子能与TfR1特异性结合，结合之后可以通过受体介导的跨胞转运机制进入细胞或穿过血脑屏障。
将小分子药与这些载体偶联可以促进许多亲水性的化疗药物或神经治疗药物进入肿瘤细胞或血脑屏障，而许多中枢神经治疗性大分子则主要通过融合蛋白的方式与抗TfR1抗体连接转运进入中枢神经系统。
Abstract：Human TfR1 was universally expressed in different tissues. The major function of TfR1 was to facilitate delivery of transferrin across cells and blood-brain barrier(BBB). As a result, iron homo-stasis was maintained. TfR1 was recognised as a critical target for tumor and brain disease therapy due to its over expression in tumor cells and BBB. In recent years, drug carriers based on TfR1 recognition were developed such as Transferrin (Tf）, anti-TfR1 antibody and TfR1 binding peptide. These carriers bind to TfR1 specifically and enter into cell or BBB through receptor mediated endocytosis. Chemicals conjugated with these carriers can be facilitated to enter into tumor cells and brain tissue. Therapeutic proteins can be engineered to fused with anti-TfR1 antibody and transported across BBB.Key words：TfR1; Tumor target therapy;Brain directed delivery1轉铁蛋白受体（TfR1)简介转铁蛋白受体（TfR1)是一种在不同组织和细胞系中普遍表达的糖蛋白。
转化生长因子-β1(TGF-β1)与肺纤维化研究的进展陈刚;余民浙【摘要】转化生长因子-β1（Transformating Growth Factorbetal，TGF-β1）是一种多功能的细胞因子，是由2条分子量为11Kd有112个氨基酸构成的单链通过二硫键结合而成的分子量为25Kd的多肽。
它在细胞的生长、分化、免疫调节、调节细胞外基质（Extracellular matrix，ECM）合成及损伤后的修复方面发挥着重要的作用。
在哺乳动物中。
TGF—β家族有3个亚型TGF—β1、TGF-β2、TGF—β3，它们通过与相应的受体结合而发挥生物作用。
活化的TGF—β过度表达对肺、【期刊名称】《中国疗养医学》【年(卷),期】2007(016)001【总页数】3页(P3-5)【关键词】转化生长因子-β1;TGF-β2;肺纤维化;细胞因子;免疫调节;细胞外基质;哺乳动物【作者】陈刚;余民浙【作者单位】066104,国家煤矿安全监察局尘肺病康复中心;066000,秦皇岛市海港医院【正文语种】中文【中图分类】R5转化生长因子－β1（Transformating Growth Factor beta1,TGF-β1）是一种多功能的细胞因子，是由 2条分子量为 11Kd有 112个氨基酸构成的单链通过二硫键结合而成的分子量为 25Kd的多肽。
它在细胞的生长、分化、免疫调节、调节细胞外基质（Extracellular matrix,ECM）合成及损伤后的修复方面发挥着重要的作用[1，2]。
在哺乳动物中，TGF-β 家族有3个亚型TGF-β1、TGF-β2、TGF-β3，它们通过与相应的受体结合而发挥生物作用。
活化的 TGF-β 过度表达对肺、肝、肾等组织病理改变的影响非常显著，特别是致纤维化方面。
在体内试验中，TGF-β1对纤维化的作用明确、TGF-β2作用不明确、TGF-β3无作用；然而体外试验发现 TGF-β 的 3个亚型都有促进纤维化的作用。
胸腺素β4研究的进展摘要：胸腺素β4是真核生物细胞中的一种主要的肌动蛋白调节因子，广泛分布于脊椎动物和无脊椎动物的多种组织中及有核细胞中。
尽管其分子水平的作用机制尚不明确，但胸腺素β4却与人类的许多生理及病理过程密切相关。
近年来对胸腺素β4的研究，发现其具有多重生物学功能，与组织再生、重塑、创伤愈合、维持肌动蛋白平衡、肿瘤发病与转移、细胞凋亡、炎症、血管生成、毛囊发育、角膜及心肌修复等密切相关。
随着研究的进一步深入，胸腺素β4在临床上的潜在应用价值将被开发，这对于一些疾病的诊断、治疗及预防均具有重要意义。
文中拟对近年来胸腺素β4在生物学功能上所取得的进展进行综述。
关键词胸腺素β4；肌动蛋白调节因子；伤口愈合；肿瘤转移；瘢痕疙瘩胸腺素(Thymosins)是由胸腺产生的一种淋巴生长因子，由Goldstein和White于1966年首次从胎牛胸腺蛋白提取液中发现，是一组小分子多肽，含有40多种组分[1]。
胸腺素根据等电点不同可分为α、β、γ三类，其中等电点位于5．0～7．0的为β族胸腺素(β-thymosins，Tβ)。
Tβ含40～44个氨基酸，结构高度保守，相对分子质量约为5000，广泛存在于脊椎动物和无脊椎动物中。
迄今已发现的Tβ成员有15个，人体内有3种，即Tβ4、Tβ10和Tβ15。
其中，胸腺素B4分布最广泛、含量最多，占β族胸腺素总量的70-80％ [2,3]。
近年来，Tβ4的生物学功能倍受人们关注，它在许多生理和病理活动中起重要作用。
研究证明，Tβ4具有多重生物学功能，与组织再生、重塑、创伤愈合、维持肌动蛋白平衡、肿瘤发病与转移、细胞凋亡、炎症、血管生成、毛囊发育、角膜及心肌修复等密切相关。
目前，人工合成的Tβ4已大量用于实验中，以探讨其在不同生理和病理活动中的作用机制。
1 Tβ4的结构与分布Tβ4首先于1981年由Low等从胸腺中分离所得，含43个氨基酸，相对分子质量为4921(乙酰化后为4963)，等电点为5.1。
肿瘤坏死因子受体超级家族（tumor necrosis fac⁃tor receptor superfamily，TNFRSF）的死亡受体（death receptor）以及它们的配体在胚胎正常发育及机体免疫和炎症反应过程中扮演了重要角色。
外胚层发育不良受体（ectodysplasin A2receptor，EDA2R）是一个在20年前被鉴定出来的TNFRSF成员（TNFRSF27）［1］，在肿瘤发生、雄激素性脱发等过程中起到重要的作用，但对于该受体作系统性介绍的综述文章尚未见报道。
本文就该受体的研究进展作一系统性的综述，旨在为相关研究提供新的思路。
1EDA2R的蛋白结构和配体1.1EDA2R的蛋白结构EDA2R基因位于人类染色体Xq12，全长约43kb，有6个外显子（GenBank登录号：NG_013271），外胚层发育不良受体EDA2R的研究进展蓝希钳1，2，肖海婷1，2，罗怀容1，2，陈建宁1，2（西南医科大学药学院：1衰老与再生医学实验室，2药理学教研室，四川泸州646000）【摘要】外胚层发育不良受体EDA2R（ectodysplasin A2receptor）是肿瘤坏死因子受体超级家族（tumor necrosis factor recep⁃tor superfamily，TNFRSF）中的一个较新的成员，在发育中的胚胎里有很高的表达，在成年人和动物的多个器官组织中也有表达。
与其它TNFRSF成员不同，尽管EDA2R蛋白在胞内没有死亡结构域（death domain，DD），但它仍可激活NF-κB和JNK通路，并介导细胞的凋亡。
本文广泛回顾了近年来与EDA2R有关的文献，就该蛋白分子的相关研究进展进行综述，以期为与该蛋白相关的分子功能或其介导的相关疾病的研究提供新的思路。
【关键词】EDA2R受体肿瘤坏死因子受体超级家族死亡结构域凋亡【中图分类号】R34文献标志码A doi：10.3969/j.issn.2096-3351.2021.03.018Research progress of ectodysplasin A2receptorLAN Xi-qian1，2，XIAO Hai-ting1，2，LUO Huai-rong1，2，CHEN Jian-ning1，2 1Key Laboratory for Aging and Regenerative Medicine；2Department of Pharmacology，School of Pharmac，South⁃west Medical University，Luzhou646000，Sichuan，China【Abstract】Ectodysplasin A2receptor（EDA2R）is a relatively new member of the tumor necrosis factor re⁃ceptor superfamily（TNFRSF），and it is highly expressed in developing embryos and is also expressed in multiple organs and tissues of adult human and animals.Different from other TNFRSF members，EDA2R protein does not contain the death domain in the intracellular region，but it can still activate the NF-κB and JNK pathways and medi⁃ate cell apoptosis.This article reviews related articles on EDA2R in recent years and related research advances in this protein，in order to provide new ideas for research on molecular functions associated with EDA2R or related dis⁃eases mediated by EDA2R.【Key words】Ectodysplasin A2receptor Tumor necrosis factor receptor superfamily Death domain Apoptosis基金项目：泸州市科技局－西南医科大学联合项目（2018LZXNYD-ZK12）；西南医科大学-泸州市中医医院基地项目（2019-LH005）第一作者简介：蓝希钳，博士。
人表皮聚角蛋白微丝蛋白的提取及其在类风湿关节炎诊断中的应用张丽君 艾脉兴 曾小峰 甘晓丹 史艳萍 宋琴芳 冷晓梅 唐福林 【摘 要】 目的 建立从人表皮提取聚角蛋白微丝蛋白(filaggrin)抗原的方法,检测抗filaggrin 抗体(AFA)在类风湿关节炎(RA)中的分布,探讨AFA对早期RA诊断的价值。
方法 从人表皮细胞中提取并部分纯化filaggrin抗原,经SDS2PA GE后用免疫印迹法检测(Western blot)103例类风湿关节炎(RA)血清和140例对照血清,包括系统性红斑狼疮(SL E)、干燥综合征(SS)、骨关节炎(OA)等。
结果 本研究方法提取的filaggrin能作为AFA免疫印迹检测的抗原。
103例RA病人中AFA敏感性和特异性分别为3519%、9317%,与疾病对照组和正常对照组相比,差异有显著性(P<0105)。
结论 从人表皮提取和部分纯化filaggrin抗原,能满足免疫印迹检测的要求。
用免疫印迹法检测RA 患者的AFA抗体,对RA有较好的敏感性和特异性,可临床用于RA的诊断。
由于基因的变异性,不同个体的filaggrin表达强弱不同。
【关键词】 多聚蛋白质类;关节炎,类风湿;印迹法,蛋白质;抗聚角蛋白微丝蛋白抗体The extraction of f ilaggrin and its signif icance in rheumatoid arthritis ZHA N G L ijun,A I M aixing, ZEN G Xiaof eng,et al.Depart ment of Rheum atology and Im m unology,Peking U nion Medical College Hospital,Beijing100730,China【Abstract】 Objective To establish the method for extracting the cytokeratin filament2aggregating protein(filaggrin)from human epidermis,to detect the anti2filaggrin antibody(AFA)in rheumatoid arthri2 tis(RA)and to determine the diagnostic value of AFA in RA.Methods Filaggrin was extracted from hu2 man epidermis and partially purified.AFA in103patients with RA and140controls was detected by West2 ern blotting.R esults Filaggrin extracted from the epidermis could be used as the antigen in AFA test by Western blotting.AFA test resulted in diagnosis of3519%of103RA samples,with a specificity of 9317%.The result was significantly higher compared with patients′and normal controls(P<0105).Con2 clusion Filaggrin extracted from the human e pidermis could be used in Western blotting.AFA has diag2 nostic value in RA.The individuality expresses heterogeneous levels of filaggrin for the gene variation.【K ey w ords】 Polyproteins;Arthritis,rheumatoid;Blotting,Western;Antifliaggrin antilbody 人表皮提取的聚角蛋白微丝蛋白(cytokeratin filament aggregating protein,filaggrin)是一种居间纤维。
介导性shRNA能抑制肺癌细胞中livin沉默基因的表达从而促进SGC-7901细胞凋亡背景—由于肿瘤细胞抑制凋亡增殖,特定凋亡的抑制因素会对于发展新的治疗策略提供一个合理途径。
Livin是一种凋亡抑制蛋白家族成员，在多种恶性肿瘤的表达中具有意义。
但是, 在有关胃癌方面没有可利用的数据。
在本研究中,我们发现livin基因在人类胃癌中的表达并调查了介导的shRNA能抑制肺癌细胞中livin沉默基因的表达，从而促进SGC-7901细胞凋亡。
方法—mRNA及蛋白质livin基因的表达用逆转录聚合酶链反应技术及西方吸干化验进行了分析。
小干扰RNA真核表达载体具体到livin基因采用基因重组、测序核酸。
然后用Lipofectamin2000转染进入SGC-7901细胞。
逆转录聚合酶链反应技术和西方吸干化验用来验证的livin基因在SGC-7901细胞中使沉默基因生效。
所得到的稳定的复制品用G418来筛选。
细胞凋亡用应用流式细胞仪(FCM)来评估。
细胞生长状态和5-FU的50%抑制浓度(IC50)和顺铂都由MTT比色法来决定。
结果—livin mRNA和蛋白质的表达检测40例中有19例(47.5%)有胃癌和SGC-7901细胞。
没有livin基因表达的是在肿瘤邻近组织和良性胃溃疡病灶。
相关发现在livin基因的表达和肿瘤的微小分化和淋巴结转移一样(P < 0.05)。
4个小干扰RNA真核表达矢量具体到基因重组的livin基因建立。
其中之一,能有效地减少livin基因的表达,抑制基因不少于70%(P < 0.01)。
重组的质粒被提取和转染到胃癌细胞。
G418筛选所得到的稳定的复制品被放大讲究。
当livin基因沉默,胃癌细胞的生殖活动明显低于对照组(P < 0.05)。
研究还表明,IC50上的5-Fu 和顺铂在胃癌细胞的治疗上是通过shRNA减少以及刺激这些细胞(5-Fu proapoptotic和顺铂)(P < 0.01)。
荧光纳米级显微镜揭示HIV-1病毒成熟过程中表面蛋白结构重排泛瑞翻译人类免疫缺陷病毒I型（Human immunodeficiency virus type 1，HIV-1）从宿主细胞出芽后依然是未成熟的粒子，其主要结构蛋白Gag经过后续的酶解后才能形成成熟的病毒粒子且具有侵染能力。
10月26日的科学杂志刊发了德国海德堡大学一个团队的研究文章，他们利用超高倍成像技术直观观察了HIV病毒粒子成熟的关键步骤。
Chojnacki等通过利用受激发射湮灭超高分辨率荧光显微镜观察HIV-1病毒外层包膜糖蛋白在病毒颗粒上的分布，发现成熟过程诱发的包膜蛋白聚集依赖于包膜蛋白尾端和Gag蛋白互作。
他们把糖蛋白在表面的聚集程度和病毒的侵染效率关联分析后发现，病毒内部和外部有着巧妙的耦合作用：内部蛋白结构的酶解后结构重排促使病毒表面结构改变，为侵染做准备。
Chojnacki等认为Gag酶解诱发的稀少的包膜蛋白二聚体在病毒表面聚集可能是HIV-1成熟的一个基本方面。
这一运用直观的高倍成像技术揭示的HIV成熟的过程和机制，有利于改善人们研发临床HIV疫苗的操作和技术，具有重要的理论和指导意义。
Chojnacki, J., T. Staudt, et al. (2012). "Maturation-Dependent HIV-1 Surface Protein Redistribution Revealed by Fluorescence Nanoscopy." Science 338(6106): 524-528.Human immunodeficiency virus type 1 (HIV-1) buds from the cell as an immature particle requiring subsequent proteolysis of the main structural polyprotein Gag for morphological maturation and infectivity. Visualization of the viral envelope (Env) glycoprotein distribution on the surface of individual HIV-1 particles with stimulated emission depletion (STED) superresolution fluorescence microscopy revealed maturation-induced clustering of Env proteins that depended on the Gag-interacting Env tail. Correlation of Env surface clustering with the viral entry efficiency revealed coupling between the viral interior and exterior: Rearrangements of the inner protein lattice facilitated the alteration of the virus surface in preparation for productive entry. We propose that Gag proteolysis-dependent clustering of the sparse Env trimers on the viral surface may be an essential aspect of HIV-1 maturation.。
Original ArticleSignal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteinsBo Wen1,2†,Yao Deng2†,Jie Guan2,Weizheng Yan2,Yue Wang2,Wenjie Tan1,2,and Jimin Gao1*1Wenzhou Medical College,Wenzhou325000,China2State Key Laboratory for Molecular Virology and Genetic Engineering,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing100052,China†These authors contributed equally to this work.*Correspondence address.Tel/Fax:þ86-10-63552140;E-mail:tanwj28@(W.T.);Tel/Fax:þ86-577-86689748;E-mail:jimingao@ (J.G.)A large number of researches focused on glycoproteins E1 and E2of hepatitis C virus(HCV)aimed at the develop-ment of anti-HCV vaccines and inhibitors.Enhancement of E1/E2expression and secretion is critical for the charac-terization of these glycoproteins and thus for subunit vaccine development.In this study,we designed and syn-thesized three signal peptide sequences based on online programs SignalP,TargetP,and PSORT,then removed and replaced the signal peptide preceding E1/E2by over-lapping the polymerase chain reaction method.We assessed the effect of this alteration on E1/E2expression and secretion in mammalian cells,using western blot analysis,dot blot,and Galanthus nivalis agglutinin lectin capture enzyme immunoassay.Replacing the peptides pre-ceding E1and E2with the signal peptides of the tissue plasminogen activator and Gaussia luciferase resulted in maximum enhancement of E1/E2expression and secretion of E1in mammalian cells,without altering glycosylation. Such an advance would help to facilitate both the research of E1/E2biology and the development of an effective HCV subunit vaccine.The strategy used in this study could be applied to the expression and production of other glyco-proteins in mammalian cell line-based systems. Keywords hepatitis C virus;signal peptide;envelope glycoprotein;expression;secretionReceived:July13,2010Accepted:October26,2010 IntroductionHepatitis C virus(HCV)is a small,enveloped, positive-stranded RNA virus that belongs to the Hepacivirus genus in the Flaviviridae family[1].HCV infection often leads to chronic hepatitis,liver cirrhosis,and hepatocellular carcinoma.Despite its clinical significance,the functional characterization of HCV glycoproteins,and thus vaccine development,has been hindered by a paucity of knowledge relating to the major structural glycoproteins E1/E2[1,2]. The HCV genome encodes a single polyprotein that is processed by cellular and viral proteases to generate10 polypeptides[3].The HCV virion is thought to have a diameter of 50nm[4].It is composed of a nucleocapsid surrounded by a host cell-derived membrane envelope that contains the viral glycoproteins E1(polyprotein residues 192–383)and E2(residues384–746)[5].Because they are exposed at the surface of the virion,the envelope pro-teins are the target of neutralizing antibodies.Therefore,E1 and E2represent the most immunologically significant HCV antigens.For this reason,E1and E2were the main focus of HCV vaccine development[4,6].HCV glycoproteins are type I membrane proteins with a C-terminal transmembrane domain(TMD)anchored in the virion phospholipid envelope.In their functional form,E1 and E2are thought to form a non-covalent heterodimer,and their TMDs are essential for heterodimerization[5,7].The ectodomains of the HCV envelope glycoproteins E1and E2 are highly modified by N-linked glycans,which have been shown to play a major role in protein folding,virus entry, and protection against neutralizing antibodies[5,7–11]. The enhancement of E1/E2expression and secretion is vital for the characterization of E1/E2structure and func-tion and for the development of an effective E1/E2-based subunit vaccine[4–7].To our knowledge,native E1/E2 expressed in mammalian cells is unsuitable for this purpose.However,some studies have shown that the optimization of signal peptide may enhance the levels of expression and secretion of these glycoproteins[12–14]. Similar strategies also have been described in researches of HIV and some other viruses[15–19].Acta Biochim Biophys Sin2011,43:96–102|ªThe Author2010.Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.DOI:10.1093/abbs/gmq117.Advance Access Publication31December2010Acta Biochim Biophys Sin(2011)|Volume43|Issue2|Page96 by guest on January 29, 2011 Downloaded fromIn this study,we constructed several plasmids encoding E1and E2to assess the effect of the replacement of the signal peptide sequences located upstream of the E1and E2genes.Our results suggested that replacing the signal sequence preceding the E1and E2genes with three signal peptide sequences significantly enhanced expression and secretion of eE1(a secreted form of E1)and eE2(a secreted form of E2)in mammalian cells.Such modifi-cations represent a significant advance in the technique available to investigate the E1and E2structure and their function and will facilitate the development of an HCV entry inhibitor and/or effective vaccine.Materials and MethodsCell culture and plasmid constructionHuman embryo kidney293T cells were grown in Dulbecco’s modified essential medium(Invitrogen, Carlsbad,USA)supplemented with10%fetal bovine serum(FBS)and100U/ml penicillin/streptomycin.Cells were routinely maintained at378C with5%CO2.Wild-type HCV1b E1and E2genes were amplified from the plasmid as described previously[20].To replace their signal peptide regions,the E1and E2genes minus the signal peptide were amplified by polymerase chain reaction(PCR)using long upstream primers.These primers were used to add the signal peptide of self-designed [21,22],tissue plasminogen activator(tPA)[23,24]or Gaussia luciferase(Gluc)[25](Table1),to the E1and E2 genes.Each of these primers contained15nucleotides of the E1or E2sequence spanning the signal peptidase clea-vage site.An Eco RV site and a Bst EII site were added in those primes for clone construction.To facilitate down-stream purification,a Bam HI restriction site and a DNA sequence encoding a His6-tag peptide were synthesized and added to the30end of the E1and E2sequences by PCR (Fig.1).Standard recombinant DNA techniques were used to generate all constructs.After purification,fragments con-taining the desired signal sequence were digested with Eco RV/Bam HI and then cloned into the same sites in the pVRC8301vector.The general scheme of plasmid con-struction is shown in Fig.1and Table2.Cell transfectionTransient transfections were performed by plating2Â105cells/well in a12-well plate12h prior to initiation of the study.When cells were 80–90%confluent,the experiments were performed.On the day of transfection,the original growth medium was replaced with a serum-free medium and DNA transfection was performed using the FUGENE HD Transfection Reagent(Roche,Basel, Switzerland)according to the standard protocols with slight modifications.Briefly,DNA solution containing1m g of pVRC-E1/E2was diluted in50m l of Opti-MEM;and the ratio of transfection reagent:DNA was6:2.Mixtures were incubated at room temperature for15min.Culture medium in12-well plates was removed and thecells were washed with Opti-MEM.A total of900m l of Table1Prediction results for different signal peptidesSignalpeptide(Sp)Sequence and cleavesite prediction aExtracellular rate ink-NN predictionSpþE1(%)SpþE2(%)E1M G C S F S I F L L AL L S C L T T P A SA ...44.4E2M V G N W A K V L IV M L L F A G V D G...55.6Self-design(sd)MD A M K V L L L VF V S P S Q V TG ...66.777.8Gluc M G V K V L F A L IC I A V A E V T G...b66.766.7tPA M D A M K R G L CC V L L L C G A V FV D S V T G ...66.766.7a Protein cleavage sites are indicated by .b The SignalP-NN result is AVA EV,but the SignalP-HMM result isVTG ,and the SIG-Pred result conforms toHMM.Figure1Schematic representation of HCV E1(A)and E2(B)expression vectors Arrows indicate signal peptide cleavage sites.Signal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin(2011)|Volume43|Issue2|Page97by guest on January 29, 2011Downloaded fromfresh serum-free Opti-MEM and50m l of transfection mixture were mixed and added to each well.After48h of incubation,the culture medium was collected and cell pellets were separated by centrifugation at1200g for 5min.Pellets were washed with PBS and then lysed in 200m l of lysis buffer for30min on ice and centrifuged at 14,500g for5min;the clarified cell lysate was collected. Clarified lysate and culture medium were used for expression analysis[20].Identical cDNA amount of pVRC was transfected as a control.PNGase F digestion for analysis of E1and E2 glycosylationsCell lysates and culture medium were diluted in sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE)loading buffer and heated at1008C for10min. PNGase F(NEB)digestion was performed in1ÂG7 buffer,with1%NP-40at378C for1h.Enzyme-treated samples were analyzed by resolving on10–13%SDS–PAGE gels and western blot analysis.Western blot analysisAfter separation by SDS–PAGE(10–13%),proteins were electroblotted onto a nitrocellulose membrane(Whatman, Florham Park,USA).Monoclonal antibodies to E1(A4)or E2(AP33)(GeneTech,Redwood,USA)were used as the primary antibody with appropriate dilution(1:1500).The horseradish peroxidase(HRP)-conjugated AffiniPure goat anti-rabbit immunoglobulin G(IgG;1:5000dilution; Sigma,St Louis,USA)was used as the secondary anti-body.Proteins were revealed on Thermo film in a dark room using the SuperSignal West Pico Chemiluminescent Substrate(Pierce,Rockford,USA)as recommended by the manufacturer.Dot blot assayA nitrocellulose membrane was pre-wetted with PBS and the Dot blot apparatus(Bio-Rad,Hercules,USA)was assembled according to the manufacturer’s introductions. Culture media and diluted cell lysates were added to wells and vacuum was applied to load products onto the mem-brane.Wells were washed twice with200m l of PBS in a low vacuum.The apparatus was disassembled and the membranes were blocked with blocking buffer(PBSþ3% FBS).After extensive washing,the primary antibody, HCV-infected human serum diluted1:200in FBS buffer, was added.Then,the secondary antibody,HRP-conjugated goat anti-mouse IgG(Sigma)diluted1:5000,was added. Galanthus nivalis agglutinin lectin capture EIA Galanthus nivalis agglutinin(GNA)lectin at1m g/ml (Sigma)was used to coat enzyme immunoassay/radio-immunoassay(EIA/RIA)plates(Costar Group,Cambridge, USA)overnight at48C.After being washed with PBS, plates were blocked with5%milk powder,and then super-natants or cell lysates were added for binding at room temperature.Plates were washed with a washing buffer (PBSþ0.05%Tween-20)four times,then100m l of primary antibody monoclonal antibody(MAbs)A4or AP33,dilution at1:1500)was added,followed by incubat-ing for90min at378C.Unbound antibody was washed off with the washing buffer.HRP-conjugated goat anti-mouse IgG(secondary antibody,100m l)was added to each well at1:5000dilution in5%milk powder and incubated for 45min at378C.Finally developed with a tetramethylbenzi-dine substrate and absorbance values at450nm were determined.ResultsReplacement of the sequence encoding the signal peptide of E1and E2The online programs SignalP,TargetP,and PSORT were used to determine cleavage sites and the expression localiz-ation of various signal peptides[21,22].Our goal was to select the peptide that could facilitate higher extracellular expression of the E1and E2proteins compared with the native signal peptide.E1and E2genes were modified to enable a series of translational fusions with different signalTable2Plasmids used in this studyPlasmid Relevant characteristicpVRC8301Efficient expression vector in eukaryotic cellsE1pVRC-E101pVRC8301derivative containing the wild-typeHCV(1b)E1(amino acids170–311)pVRC-E102pVRC-E101derivative with the signal peptidesequence replaced with a self-designed signalsequencepVRC-E103pVRC-E101derivative with the signal peptidesequence replaced by that of tPApVRC-E103pVRC-E101derivative with the signal peptidesequence replaced by that of GlucE2pVRC-E201pVRC8301derivative containing the wild-typeHCV(1b)E2(amino acids364–661)pVRC-E202pVRC-E201derivative with the signal peptidesequence replaced with a self-designed signalsequencepVRC-E203pVRC-E201derivative with the signal peptidesequence replaced by that of tPApVRC-E203pVRC-E201derivative with the signal peptidesequence replaced by that of GlucSignal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin(2011)|Volume43|Issue2|Page98 by guest on January 29, 2011 Downloaded frompeptide-encoding regions.An Eco RV site and a Bst EII site were inserted at each end of the signal peptide-encoding region(Fig.1).The Bst EII insertion created a mutation in the C-terminal of each signal peptide,in which the terminal amino acid was replaced with V-T-G(Fig.1).Software analysis indicated that these replacements did not alter the cleavage site of E1/E2(Table1).For expression,the frag-ment obtained by digesting each PCR product with Eco RV/Bam HI was inserted at the same sites in the pVRC8301vector.Plasmids containing the wild-type signal peptide sequence were named pVRC-E101and pVRC-E201;plasmids containing the computer-designed signal peptide sequence were named pVRC-E102and pVRC-E202;plasmids containing the Gluc signal peptide sequence were named pVRC-E103and pVRC-E203;and plasmids containing the tPA signal peptide sequence were named pVRC-E104and pVRC-E204(Table2).In each case,successful insertion was confirmed by sequencing. Effects of signal peptide replacement on intracellular expression of E1and E2The signal peptide sequence of the HCV E1/E2glyco-proteins was substituted with three heterologous signal peptide sequences.Intracellular and extracellular expression levels of E1and E2proteins were assessed using a mono-clonal antibody specific for either E1or pared with the control(pVRC-E101/pVRC-E201),all of the clones (pVRC-E102/pVRC-E202,pVRC-E103/pVRC-E203,and pVRC-E104/pVRC-E204)successfully expressed the E1or E2glycoproteins.HCV E1expressed in untreated cells had an apparent molecular mass of33kDa and the molecular weight(MW)was reduced to18kDa after deglycosylation, and the MW of E2is60–70kDa in nature and31kDa after deglycosylation[3].Our results showed that the MW of each modified signal peptide attached to E1and E2did not differ significantly from the wild type(Fig.2).Furthermore, after digestion with N-glycosidase F,the MWs of E1and E2with the modified signal peptide were not significantly different from that of wild type,suggesting a pattern of identical glycosylation(Fig.3).Native E1glycoprotein was detected with the least expression,despite its expression conditions was identical with others.Only by increasing the loading quantity to 30m g(six times of the loading quantity than that of other samples),the band of native E1glycoprotein can be detected(Fig.2).However,band intensity remained notice-ably lower than any of the modified E1proteins.Thus,pro-duction of E1with a wild-type signal sequence was particularly low,at least less than one-sixth,that of E1 with either the tPA or Gluc signal peptide.Effect of signal peptide replacement on secretionof E1/E2After confirming the intracellular expression of E1/E2with the replaced signal peptide,we assessed the extracellular concentrations of the E1and E2proteins.As predicted by software,the ratios of secretion were different among the various signal peptides.To assess the true expression level of the modified E1and E2glycoproteins,we chose methods that could enrich the non-denature productions for more sen-sitive detection,such as dot blot and GNA capture EIA, both the semi-quantitative methods.They were used to detect glycoproteins E1and E2in all cell lysates and culture media in parallel.Results from these two methods showed no obvious disparities(Fig.4).The expression level of the E1native signal peptide was particularly low,and three of the modified signal peptides,particularly tPA and Gluc, showed significantly increased expression than thenative Figure2Western blot analysis of E1/E2produced by293T transfected with different plasmids Cell lysate samples(L,5m g/sample)and supernatants(S,20m g/sample)were electrophoresed separately.All samples were collected after48h cultivation with non-BSA opti-MEM.The blot was stripped and re-probed with b-actin.Asterisk denotes that the loading volume of this sample was six times greater(30m g)than that of other lysate samples.293T cells transfected without a plasmid was used as a control(mock).Signal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin(2011)|Volume43|Issue2|Page99 by guest on January 29, 2011 Downloaded fromsignal peptide.Such distinction was not so obvious in E2,but the expression level of HCV E2glycoprotein with a native signal peptide was still lower than that of E2modified with any of the exogenous signal peptides.Otherwise,the expression level of pVRC-E102/pVRC-E202,whose signal peptide was predicted to have the greatest secretion ability,was lower than the sample with tPA or Gluc signal peptides.Furthermore,modified with the tPA or Gluc signal peptides,neither the expression nor the secretion levels of glyco-proteins showed any marked differences (Fig.4).In our experiment,E2could be more easily detected than E1.This might be caused by different primary antibody,or because E1/E2were glycosylated at different levels leading to dis-tinct GNA-binding ability.DiscussionMore than 120million people worldwide are chronically infected with HCV,making HCV infection to be the leading cause of liver transplantation in developed countries.Treatment options are limited,and the efficacy depends on both the infecting strain and the initial viral load [1].During the translation of HCV,the nascent E1and E2polypeptides are targeted to the host endoplasmic reticulum membrane for modification by N-linked glycosylation [5].E1and E2are released from the polyprotein through cleavage by a host signal peptidase [3]and are anchored in the viral lipid envel-ope as a heterodimer,which plays a major role in the HCV entry [7,11].Deletion of the TMDs of E1and E2results in the secretion of these truncated forms of HCV glycoproteins into the extracellular medium.E2antigen in itssecretedFigure 4Analysis of the production levels of E1and E2glycoproteins containing altered signal peptides Human embryo kidney 293T cells contained the following plasmids were indicated,and 293T cells transfected without a plasmid was used as a control (mock).Cell lysates (L)and supernatants (S)were collected 48h after transfection.(A)and (B)represent the dot blot results of E1and E2glycoproteins detected using HCV-infected human serum as the primary antibody.GNA lectin capture EIA data are shown in (C)and (D).Data obtained from both the dot blot and EIA methods showed no markeddiscrepancies.Figure 3Different signal peptide have no apparently effect on the expression of E1and E2in 293T cells 293T cells contain pVRC-E101/104and pVRC-E201/204plasmids were collected 48h after transfection.Cell lysate (L)and supernatant (S)of each samples were treated with PNGase F (W)or left untreated (W/O).After being separated by SDS–PAGE (E1in 13%and E2in 10%acrylamide)under reducing conditions,E1and E2were immunoprecipitated with monoclonal antibodies A4and AP33.(A)In the same loading quantity (20m g),bands detected from E101(arrow indict)were much fainter than those of E104,but their distributions were the same under the similar treatment.(B)The distinction of band grayscale between E201and E204is not obvious,but the MW and distribution of their bands are identical under the same treatment.Signal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin (2011)|Volume 43|Issue 2|Page 100by guest on January 29, 2011 Downloaded fromform leads to an increased humoral response in mice[26]. HCV E1and E2are primary determinants of entry and pathogenicity.HCV E2glycoproteins are involved in recep-tor binding,virus–cell fusion,and entry into host cells[11]. However,its role in membrane fusion and immune evasion remains uncharacterized.The function of E1is unknown; however,it is a target for neutralizing antibodies and its association with E2is essential for viral entry[11]. Increasing knowledge of the nature and the function of HCV E1and E2glycoproteins help to develop antiviral drugs and vaccine candidates.The production of large quantities of functional and secreted E1and E2proteins will allow us to perform compre-hensive biochemical and biophysical analysis,which will lead to the development of a vaccine that is effective against HCV infection.In this study,we developed a novel expression system for producing the secreted form of E1(eE1)and E2 (eE2)ectodomains from mammalian cells via a signal peptide sequence replacement strategy and performed a comprehen-sive biochemical characterization.These data enhance our understanding of HCV envelope glycoproteins and may assist in the design of HCV vaccines and entry inhibitors. Our data showed that the replacement of the signal peptide sequences located at the upstream of E1and E2 genes altered their secretion and expression levels,and most importantly,the replacement did not affect glycosyla-tion.Furthermore,replacing them with tPA and Gluc resulted in the greatest increases at expression and secretion levels of eE1and eE2in mammalian cells.We believed that the strategy used in this study could be applied to the expression and production of other glycoproteins in mam-malian cell line-based systems.The next challenge is to use our novel strategy to gener-ate large quantities of HCV E1and E2envelope glyco-proteins.This study will allow us to elucidate their structural biology and the mechanism(s)involved in the interactions between these glycoproteins and other cellular factors,eventually to facilitate the development of an HCV vaccine and/or entry inhibitor. AcknowledgementsThe authors thank Dr Gary Nabel(Vaccine Research Center,National Institute of Allergy and Infectious Diseases,National Institutes of Health,Bethesda,USA)for the pVRC plasmid and Dr Jean Dubuisson(Universite´Lille Nord de France,CNRS-UMR8161,Institut Pasteur de Lille,Lille,France)for the A4antibody.FundingThis work was supported by the grants from the Hi-Tech Research and Development Program of China(2007AA02Z455,2007AA02Z157)and the State KeyLaboratory for Molecular Virology and Genetic Engineering. References1Lindenbach BD,Thiel HJ and Rice CM.Flaviviridae:the viruses and theirreplication.In:Knipe DM and Howley PM eds.Fields Virology.Philadelphia,PA:Lippincott Williams&Wilkins,2007,1101–1152.2Houghton M and Abrignani S.Prospects for a vaccine against the hepatitisC virus.Nature2005,436:961–966.3Dubuisson J.Hepatitis C virus proteins.World J Gastroenterol2007,13:2406–2415.4Wakita T,Pietschmann T,Kato T,Date T,Miyamoto M,Zhao Z andMurthy K,et al.Production of infectious hepatitis C virus in tissue culturefrom a cloned viral genome.Nat Med2005,11:791–796.5Lavie M,Goffard A and Dubuisson J.Assembly of a functional HCV gly-coprotein heterodimer.Curr Issues Mol Biol2007,9:71–86.6Stamataki Z,Coates S,Evans MJ,Wininger M,Crawford K,Dong C andFong YL,et al.Hepatitis C virus envelope glycoprotein immunization ofrodents elicits cross-reactive neutralizing antibodies.Vaccine2007,25:7773–7784.7Ciczora Y,Callens N,Penin F,Pecheur EI and Dubuisson J.Transmembrane domains of hepatitis C virus envelope glycoproteins:resi-dues involved in E1/E2heterodimerization and involvement of thesedomains in virus entry.J Virol2007,81:2372–2381.8Drummer HE,Maerz A and Poumbourios P.Cell surface expression offunctional hepatitis C virus E1and E2glycoproteins.FEBS Lett2003,546:385–390.9Falkowska E,Kajumo F,Garcia E,Reinus J and Dragic T.Hepatitis Cvirus envelope glycoprotein E2glycans modulate entry,CD81binding,and neutralization.J Virol2007,81:8072–8079.10Helle F,Goffard A,Morel V,Duverlie G,McKeating J,Keck ZY and Foung S,et al.The neutralizing activity of anti-hepatitis C virus antibodiesis modulated by specific glycans on the E2envelope protein.J Virol2007,81:8101–8111.11Cocquerel L,Voisset C and Dubuisson J.Hepatitis C virus entry:potential receptors and their biological functions.J Gen Virol2006,87:1075–1085.12Martoglio B and Dobberstein B.Signal sequences:more than just greasy peptides.Trends Cell Biol1998,8:410–415.13Futatsumori-Sugai M and Tsumoto K.Signal peptide design for improving recombinant protein secretion in the baculovirus expression vector system.Biochem Biophys Res Commun2010,391:931–935.14Zhang L,Leng Q and Mixson AJ.Alteration in the IL-2signal peptide affects secretion of proteins in vitro and in vivo.J Gene Med2005,7:354–365.15Li Y,Luo L,Thomas DY and Kang CY.The HIV-1Env protein signal sequence retards its cleavage and down-regulates the glycoprotein folding.Virology2000,272:417–428.16Bruno M,Roland G and Bernhard D.Signal peptide fragments of prepro-lactin and HIV-1p-gp160interact with calmodulin.EMBO J1997,16:6636–6645.17Robert E,Oliver L,Thomas S,Markus E,Hans-Dieter K and WolfgangG.Identification of Lassa virus glycoprotein signal peptide as a trans-acting maturation factor.EMBO Rep2003,4:1084–1088.18Lobigs M,Zhao HX and Garoff H.Function of Semliki forest virus E3 peptide in virus assembly:replacement of E3with an artificial signalpeptide abolishes spike heterodimerization and surface expression of E1.J Virol1990,64:4346–4355.19Paul AR,Mark LH,Toni W,Helen LR and Alan P.Antigenic and genetic characterization of the haemagglutinins of recent cocirculating strains ofinfluenza B virus K.J General Virol1992,73:2737–2742.Signal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin(2011)|Volume43|Issue2|Page101by guest on January 29, 2011Downloaded from20Bian T,Zhou Y,Bi S,Tan W and Wang Y.HCV envelope protein func-tion is dependent on the peptides preceding the glycoproteins.Biochem Biophys Res Commun2009,378:118–122.21Nakai K and Horton P.PSORT:a program for detecting sorting signals in proteins and predicting their subcellular localization.Trends Biochem Sci 1999,24:34–36.22Emanuelsson O,Brunak S,von Heijne G and Nielsen H.Locating proteins in the cell using TargetP,SignalP and related tools.Nat Protoc2007,2: 953–971.23Pennica D,Holmes WE,Kohr WJ,Harkins RN,Vehar GA,Ward CA and Bennett WF,et al.Cloning and expression of human tissue-type plasmino-gen activator cDNA in E.coli.Nature1983,301:214–221.24Qiu JT,Liu B,Tian C,Pavlakis GN and Yu XF.Enhancement of primary and secondary cellular immune responses against human immunodefi-ciency virus type1gag by using DNA expression vectors that target Gag antigen to the secretory pathway.J Virol2000,74:5997–6005.25Knappskog S,Ravneberg H,Gjerdrum C,Tro¨sse C,Stern B and Pryme IF.The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide.J Biotechnol2007,128:705–715.26Abraham JD,Himoudi N,Kien F,Berland JL,Codran A,Bartosch B and Baumert T,et parative immunogenicity analysis of modified vacci-nia Ankara vectors expressing native or modified forms of hepatitis C virus E1and E2glycoproteins.Vaccine2004,28:3917–3928.Signal peptide replacements enhance HCV envelope glycoprotein expression and secretionActa Biochim Biophys Sin(2011)|Volume43|Issue2|Page102 by guest on January 29, 2011 Downloaded from。
1周博达1 侯超1 综述 王月丹1北京大学医学部基础医学院 100083Email: wangyuedan@摘要: 血管内皮生长因子通过与其受体结合发挥生物学作用，促进内皮细胞有丝分裂及其增殖分化、迁移，导致血管新生，形成血管翳，在类风湿关节炎的病理过程中发挥关键的作用。
对抗血管内皮生长因子及其受体，从而阻止新生血管的形成为治愈类风湿关节炎提供了新的途径。
关键词：类风湿性关节炎；血管内皮生长因子；受体；类风湿关节炎(rheumatoid arthritis, RA)是一种严重的自身免疫功能障碍性疾病，在世界范围内的平均患病率约1%，而我国约有400 万RA患者。
其临床表现以反复发作的多发性小关节炎和不同程度侵蚀性改变及进行性强硬和畸形为主，并有骨和骨骼肌萎缩，是一种致残率较高的疾病。
RA的病理特征为关节滑膜慢性炎症、软骨吸收、骨质破坏和骨纤维化，其中一个重要的病理生理过程是新生血管翳的形成，而新生血管的生成是产生和维持RA血管翳的重要标志。
在RA发展过程中，滑膜组织中存在大量的血管,出现了血源性细胞的浸润、增殖和水肿，在血管内皮细胞里层也出现了对细胞因子等作用的靶点，同时内皮细胞对这些因子的反应可以维持和促进RA病理过程的发展，特别是新血管的生成[1]。
近年来，人们已经认识到血管生成在RA的侵蚀和破坏过程中发挥了重要作用。
中断血管生成在类风湿性关节炎的治疗中有重要的意义。
RA患者的关节滑膜中，存在一些生长因子、细胞因子和化学增活素从而影响血管的生成，许多生物活性物质还能通过上调血管生成刺激素的表达从而发挥间接作用。
其中，血管内皮生长因子及其受体在血管新生过程中起着重要的作用。
一、关于血管内皮生长因子及其受体血管内皮生长因子(vascular endothelial growth factor，VEGF)，又称为血管通透性因子(vascular permeability factor, VPF)，于1989年由Ferrara等发现，属于血小板源生长因子(platelet-derived growth factor, PDGF) 家族成员，是一种相对分子量在34000-46000的同源二聚体糖蛋白，由两个亚基通过二硫键结合，其热稳定性高，序列高度保守。
In vitro evolution of an antibody fragment population tofind high-affinity hapten bindersHelena Persson1,Henrik Wallmark1,Anne Ljungars2, Johan Hallborn2and Mats Ohlin1,31Department of Immunotechnology,Lund University,BMC D13SE-22184, Lund and2BioInvent International AB,So¨lvegatan41SE-22370,Lund, Sweden3To whom correspondence should be addressed.E-mail:mats.ohlin@immun.lth.seRecently,we constructed a focused antibody library tai-lored to interact with haptens.High functionality of this library was demonstrated,as specific binders could be retrieved to a range of different haptens.In the current study we have developed a mutagenesis and selection strategy in order to furtherfine-tune the hapten binding properties of these antibody fragments.Testosterone was chosen as model antigen for the investigation.A popu-lation,rather than a single clone,originating from this focused library and enriched for testosterone binders, was subjected to random mutagenesis and different phage display selection strategies of various stringencies. These included consecutively lowering the antigen con-centration and having,or not having,soluble hapten present during the phage capture and elution steps.The different selection procedures resulted in a considerable increase in apparent affinities for several of the selected populations,from which the highest affinity antibody iso-lated had a K D of2nM,corresponding to an 200-fold affinity improvement compared with the best clone of the starting population.Importantly,the polyclonal nature of the starting material allowed for the identification of novel unrelated variants that differed infine-specificity, demonstrating that this approach is valuable for explor-ing different parts of structure space.Keywords:affinity maturation/antibody library/hapten/phage display/testosteroneIntroductionBesides their importance in the adaptive immune response, antibodies have emerged as an invaluable tool in applications ranging from basic research to disease diagnostics and therapy.The hybridoma technology(Ko¨hler and Milstein, 1975),which relies on animal immunizations,is still the most common way of obtaining antibodies,in particular for diagnostic applications.However,within the past two decades,since the cloning of thefirst antibody repertoires (Huse et al.,1989;Ward et al.,1989)and with the successful production of antibody fragments in bacteria(Skerra and Plu¨ckthun,1988)combined with the functionally display of these on phage(McCafferty et al.,1990;Kang et al.,1991), antibody library technology has gradually become the method of choice for the development of specific binders. Over the years numerous antibody libraries have been reported(So¨derlind et al.,2001;Hoogenboom,2002).Various strategies,using natural as well as synthetic diversity,have been utilized,often with the goal to create very large libraries with diversity high enough to provide binding specificity against any antigen.Although in many cases successful,it has,despite the size of these so called universal or single-pot libraries,been difficult to isolate high-affinity antibodies to some particularly troublesome antigens.In specific appli-cations,libraries tailored to interact with a certain antigen or group of antigens,so called focused libraries,have therefore been proposed as an attractive alternative(Collis et al.,2003; Almagro,2004;Almagro et al.,2006;Kehoe et al.,2006; Sidhu and Fellouse,2006).Recently,we successfully con-structed such a focused library with features biased to recog-nize haptens(Persson et al.,2006).The main principle behind the design of the focused hapten library was based on the fact that the topography of the binding site correlates with the size of its antigen (Webster et al.,1994;MacCallum et al.,1996).As small-molecule antigens almost exclusively interact with cavity-shaped paratopes,a hapten-specific antibody,displaying a pronounced binding pocket,was used as structural backbone for library construction.By keeping framework and canoni-cal structure determining residues constant,we reasoned that a large portion of the library would maintain the cavity topo-graphy as seen in the wild-type ing site-directed mutagenesis,diversity was mainly restricted to positions and residue types that are commonly involved in hapten contacts (MacCallum et al.,1996;Almagro,2004),resulting in vari-ation of mainly centrally located cavity-lining residues.To possibly allow for the formation of cavities of variable depth (Collis et al.,2003),some variation in loop length was also introduced.Despite the relatively small size of this so-called cavity library(5Â107),we were able to identify highly specific antibodies to a range of different haptens. Importantly,we could show that the cavity library was a superior source of hapten-binders compared with a consider-ably larger general-purpose repertoire(Persson et al.,2006). In order to be generally applicable,a library has to be able to provide binders of high specificity as well as of high affi-nity.However,as no particular efforts were made to select for binding strength in the initial study(Persson et al.,2006), the affinity of the obtained hapten-binders were relatively modest(K D¼1026–1027M).Therefore,in the present investigation we set out to further explore the evolution potential of the cavity library to deliver hapten-binding anti-bodies with affinities required for practical applications. MethodsConstruction of librariesThe starting material for library construction was based on a sub-population of the previously described cavity library (Persson et al.,2006).This library had been selected twice on testosterone-biotin(testosterone3-(5-(6-(5-(biotin) pentanamido)hexanamido)pentylcarbamoyl)propanoate) coupled to avidin-coated magnetic beads(Persson#The Author2008.Published by Oxford University Press.All rights reserved. For Permissions,please e-mail:journals.permissions@ 485Protein Engineering,Design&Selection vol.21no.8pp.485–493,2008Published online May13,2008doi:10.1093/protein/gzn024at Huazhong Agricultural University on January 26, Downloaded fromet al .,2006).Plasmid DNA was isolated from this population,from here on designated CT (C for cavity library and T for testosterone),and submitted to one or two rounds of random mutagenesis by error-prone PCR using an excess of guanine and/or thymine (Fig.1).In the first round,the reaction mix (100m l)contained 10mM Tris–HCl (pH 8.3),50mM KCl,9mM MgCl 2,0.75mM MnCl 2,0.001%gelatin (w/v),0.75mM dNTP and an additional 3.2mM dGTP or dTTP,5U AmpliTaq DNA polymerase (Applied Biosystems,Foster City,CA,USA),0.4m M each of the vector-specific primers GGCCGATTCATTAATGCAGC and GCCTTTAGCGTCAG ACTGTAG and as template 10ng of the purified vector was used.The DNA was amplified for 26cycles (948C 1min,568C 1min,728C 2min).The two different PCR products,resulting from either an excess in dGTP (denoted CTep1)or dTTP,were pooled and used as a template in the second PCR.The same conditions as for the first round were used,however,only an excess of dGTP was used this time,creat-ing the library CTep2.The mutated gene fragments CTep1and CTep2were gel purified and digested with SfiI and Not I (New England Biolabs,Beverly,MA,USA)followed by lig-ation into the corresponding sites of a modified version of the pFab5c.His phagemid (Engberg et al .,1995),shortened to encode only the final C-terminal domain of protein III.The libraries were subsequently transformed into electrocom-petent Top10F’Escherichia coli (Invitrogen,Carlsbad,CA,USA)and random clones sequenced from both libraries.Mutational rates were determined by calculating the percen-tage of mutated residues at the nucleotide or amino acid level,excluding those positions that were targeted for diver-sification in the cavity library [38H ,40H ,55H ,57H ,59H ,66H ,107H -109H ,113H ,40L ,105-117L ;the subscript relates to its position in the heavy (H)or light chain (L)](Persson et al .,2006).Throughout this study,residue numbering follows the IMGT nomenclature (Lefranc et al .,2003),inserting gaps at the top of the loops.The three loops of the heavy and light chain are denoted complementarity determining region (CDR)H1-3and CDRL1-3,respectively.Phage selectionsPhage stocks of the two libraries and CT were prepared by VCSM13helper phage infection (Stratagene,La Jolla,CA,USA)as described (Cicortas Gunnarsson et al .,2004).An overview of the selection procedure is illustrated in Fig.1and Table I.Briefly,three selection rounds were performed,using gradually lower concentration of testosterone-biotin (Sigma-Aldrich Inc.,St Louis,MO,USA)(Fig.2).Also between the rounds,the coating of the paramagnetic beads was alternated between streptavidin (Dynal A/S,Oslo,Norway)and avidin (Spherotech Inc.,Libertyville,IL,USA)to reduce the risk of obtaining carrier-specific binders.In the first round,the three different libraries (CT,CTep1and CTep2)were handled separately.The libraries were pre-incubated with testosterone-biotin diluted in selection buffer [0.2%gelatin,0.05%(v/v)Tween 20in phosphate-buffer saline (PBS)]for 2h on rotation at room temperature.Magnetic beads,washed twice in selection buffer,were added and the mixture was allowed to incubate for another 30min.After five washes in selection buffer,followed by two washes in PBS,binding phages were eluted by the addition of trypsin (Invitrogen,Pasley,UK),which cleaves at a site located between the displayed single chain antibody fragment (scFv)and protein III.The cleavage reaction was subsequently inhib-ited by the addition of aprotinin (Invitrogen).Eluted phages were propagated and titers determined as previously described (Cicortas Gunnarsson et al .,2004).Equal amounts of phages from the three selections were pooled and used for the second round of selection,which was carried out essentially as described for the first round but with the following changes;the amount of panning antigen was decreased (from 5Â1028to 1Â1028M)and the phage capture step was varied,by excluding or including soluble testosterone (1026M,Sigma-Aldrich)as competitor at the time of addition of the magnetic beads.The two resulting populations (C2L and C2H )were treated separately in the third selection round.In addition to varying the antigen concentrations and conditions during the capturing step,the elution method was also altered,yield-ing in total eight different selection strategies (Table I).The strategies were named with three letters;A,C and E,coding for antigen concentration,capture condition and elution method,respectively.The letters were indexed with high (H)or low (L)to denote the selection pressure for each variable.A H for the lower antigen concentration (2Â1029M),A L for the higher (1028M),C H if soluble testosterone (1026M)wasFig.1.A flowchart outlining the experimental steps of the study.The starting material of the affinity maturation process (library CT)was a phage-selected sub-population of the previously described cavity library (Persson et al .,2006).Library CT,which was highly enriched for testosterone binders,was submitted to one or two rounds of random mutagenesis by error-prone PCR,resulting in the two libraries CTep1and CTep2.Different phage display selection strategies were subsequently used on these libraries to find antibody fragments of increased affinity (for further details on selection parameters see Table I).After a first round of selection,in which the three starting libraries (CT,CTep1and CTep2)had been handled separately,the libraries were pooled and used for a second round of selection.By varying the conditions during the phage capture step,two populations (C2L and C2H )were obtained.These were treated separately in the third selection round,giving in total 8different selection strategies.These were named with three letters;A,C and E,coding for Antigen concentration,Capture condition and Elution method,respectively,and furthermore indexed with H (high)or L (low)to denote selection pressure for each variable.All 16populations depicted were analyzed by competitive phage ELISA and individual scFv obtained from the 10populations of selection rounds two and three were also assayed in soluble format (for further details see Table II).H.Persson et al.486at Huazhong Agricultural University on January 26, 2011 Downloaded frompresent during the capturing step and C L if it was not,E L if the phages were eluted with trypsin or E H if eluted with soluble testosterone (1025M,2h).In all three selection rounds an excess of magnetic beads were used to allow all antigen-bound phages to be captured and to minimize the risk of selecting multivalently displaying phages that otherwise,due to avidity effects,most likely would have a selection advantage.Phage ELISA and sequencing of selected populationsThe unselected libraries (CT,CTep1and CTep2)as well as the different populations from the three selection rounds were analyzed for specificity using phage ELISA.The popu-lations were tested on testosterone-biotin and FITC-biotin (5(6)-(biotinamidohexanoylamido)pentylthioureidylfluores-cein)(Sigma-Aldrich),both bound to the plate via streptavi-din,and on bovine serum albumin.Antigen-bound phages were detected by a horseradish peroxidase (HRP)-conjugated anti-M13antibody (GE Healthcare,Uppsala,Sweden)using o -phenylenediamine as chromogen.In addition,six clones,randomly picked from each of the eight strategies of the third selection round,were similarly analyzed for specificity and subsequently sent for automated DNA sequencing (MWG Biotech,Ebersberg,Germany).To estimate the binding affinity for soluble testosterone,the different populations were further analyzed in a competi-tive phage ELISA format.Testosterone was used to compete with testosterone-biotin immobilized on streptavidin as described (Persson et al .,2006).This allowed for the deter-mination of IC 50values,i.e.the concentration of competing hapten that inhibited 50%of phages binding to the immobi-lized antigen.IC 50values were similarly determined for estradiol (Sigma-Aldrich),fluorescein (Sigma-Aldrich)and testosterone-biotin.Before the addition of the phage solution containing the inhibiting antigen (1–20000ng/ml),care was taken to saturate the biotin-binding sites of streptavidin by adding an excess of testosterone-biotin,a crucial step when inhibiting with biotinylated substances as in the case of testosterone-biotin.For comparison studies,two testosterone-specific clones (TC-5and TC-20,GenBank accession numbers DQ250208and DQ250223,respectively),which previously had been isolated from the cavity library (Persson et al .,2006),were alsoanalyzed.Fig.2.Molecular structures of testosterone-biotin (A),the derivative used for the phage display selections and the subsequent ELISA and BIAcore assays,and testosterone (B).T a b l e I .O v e r v i e w o f t h e s e l e c t i o n s t r a t e g i e sS e l e c t i o n 1S e l e c t i o n 2S e l e c t i o n 3I n p u t A n t i g e nC a t c h i n g E l u t i o nO u t p u tI n p u tA n t i g e n C a t c h i n gE l u t i o nO u t p u tI n p u t A n t i g e nC a t c h i n g E l u t i o n O u t p u tC T5*1028M T BS A T r y p s i n C T *C T *C T e p 1*C T e p 2*p o o l e d t o g e t h e r1028M T BA v i d i nT r y p s i nC 2LC 2L2*1029M T B S A T r y p s i n A H C L E LC 2L2*1029M T B S A 1025M T A H C L E HC T e p 15*1028M T BS AT r y p s i n C T e p 1*C 2L1028M T B S A T r y p s i n A L C L E LC 2L1028M T B S A 1025M T A L C L E HC T *C T e p 1*C T e p 2*p o o l e d t o g e t h e r 1028M T BA v i d i n þ1026M TT r y p s i nC 2HC 2H2*1029M T B S A þ1026M T T r y p s i n A H C H E LC 2H2*1029M T B S A þ1026M T 1025M T A H C H E HC T e p 25*1028M T B S AT r y p s i n C T e p 2*C 2H1028M T B S A þ1026M T T r y p s i n A L C H E LC 2H1028M T BS A þ1026M T 1025M TA L C H E H TB ,t e s t o s t e r o n e -b i o t i n .S A ,s t r e p t a v i d i n .T ,t e s t o s t e r o n e .In vitro evolution of an antibody fragment487at Huazhong Agricultural University on January 26, 2011 Downloaded fromHigh-throughput ELISA screeningFor the subsequent analysis steps,an automated ELISA screening procedure was performed using integrated robotic workstations as reviewed by Hallborn and Carlsson(2002). For this purpose a change to a compatible vector,that allows the production of soluble scFv,was needed.Phagemide DNA from the ten populations of selection rounds two and three as well as from the control clones TC-5and TC-20was isolated and digested with the restriction enzymes SfiI and Avr II(New England Biolabs).The fragment encoding the scFv was gel-purified and subsequently inserted into a vector that provides the secreted scFv with three FLAG-epitopes and a hexahistidine tag at their C-termini(BioInvent International AB,Lund,Sweden).The constructs were trans-formed into chemically competent Top10E.coli using stan-dard heat chock treatment.A total of1152clones from each strategy were picked, transferred to384-well plates containing media supplemented with isopropyl thiogalactoside and scFv were produced over-night at378C with vigorous shaking.After sedimentation of the bacteria,the supernatants containing the scFv were screened for binding to biotinylated testosterone in a primary and a secondary assay.Briefly,the produced scFv were cap-tured by anti-FLAG antibodies(M2,Sigma-Aldrich)that had been adsorbed on the surface of microtiter plates.By limiting the anti-FLAG antibody,differences in scFv production levels should minimally affect the assay,making any observed differences in signal dependent on binding ability. Following a washing-step,testosterone-biotin was added (40nM).After1h of incubation,unbound antigen was removed and HRP-labeled streptavidin(Dako Cytomation, Glostrup,Denmark)added.After a last washing-step,lumi-nescent substrate(Pierce supersignal,Pierce,Rockford,IL, USA)was added and the plates were read in a Tecan Ultra instrument(Tecan AG,Ma¨nnedorf,Switzerland).To get a value of the binding properties of the scFv,a threshold was set using the clones TC-5and TC-20with known affinity (Persson et al.,2006).FITC-biotin was used as a negative reference to assess the specificity of the interaction.The48 clones from each strategy that gave the highest specific signals in the primary assay were selected for a secondary analysis.The basic set-up of both screenings was the same; however,the secondary assay was much more stringent, using lower testosterone-biotin concentration(10nM)and an increased number of washes.From the secondary screen-ing twelve clones were picked for sequencing from each selection strategy,thefive clones that gave the highest signal and seven clones picked at random.Of the96sequenced clones,nine unique clones were identified(designated TM-1–9).Competitive immunoassays of soluble scFvTo investigate the binding affinity of the nine unique clones for testosterone and testosterone-biotin,competitive ELISA on crude expression supernatants was performed,essentially as described for the phage-displayed scFv but with the following changes:scFv fragments were detected using the HRP-labeled anti-FLAG M2antibody(Sigma-Aldrich)and the reaction developed using luminescent substrate.The binding to estradiol andfluorescein was also accessed.The concentration range used for the inhibiting antigens were0.05–20000ng/ml.The ELISA IC50values were subsequently verified in a competitive BIAcore assay using a BIAcore3000instrument (BIAcore AB,Uppsala,Sweden).Streptavidin was covalently coupled to a CM5sensor chip using amine coupling accord-ing to the manufacturer’s instructions and an excess of testosterone-biotin,enough to cover all available biotin-binding sites,was added.A secondflow cell was treated with the same chemical procedure but without the addition of antigen and used as reference.Afixed concentration of scFv (TM-2,TM-5,TM-7,TC-5and TC-20),purified from the periplasmic space using affinity chromatography on Ni-NTA agarose(Qiagen,Hilden,Germany),was mixed with a range of concentrations(0–20000ng/ml)of the inhibiting antigens (testosterone,testosterone-biotin,estradiol andfluorescein), allowed to reach equilibrium and subsequently injected onto the chip at aflow rate of20m l/min.All experiments were performed at258C and the chip was regenerated in20mM HCl,150mM NaCl.Response curves were generated by sub-tracting the signal obtained from the controlflow cell and IC50values determined.Of note,none of the analyzed scFv showed any signs of reactivity with streptavidin.ResultsLibrary construction and analysisThe aim of the study was to develop a mutagenesis and selection system(Fig.1)tofind high-affinity binders for haptens,in this study exemplified by testosterone.The CT library,a sub-population of the previously described cavity library(Persson et al.,2006),was chosen as our initial source of diversity.The cavity library had been designed to include binders of high frequency and functionality against a broad range of different haptens.This library had been sub-jected to two rounds of phage selections on testosterone, yielding the CT library.Analysis of individual members of this pool by ELISA showed that$90%of these were testos-terone specific.Also,sequencing revealed a high diversity among those residues targeted for modification(Persson et al.,2006),providing a polyclonal nature of the starting material used for evolution.Additional diversity was introduced randomly by error-prone PCR(Fig.1).In this way the two libraries CTep1and CTep2were created,each having an estimated size of3–4Â107clones of which89%had an insert and80%were in-frame(data not shown).If excluding those positions tar-geted in the design of the cavity library,the random nucleo-tide substitution frequencies of CTep1and CTep2were0.77 and0.87%,respectively,giving an average of3.8(range1–6)and3.9(range2–7)amino acid substitutions per scFv (data not shown).This procedure resulted in$6–8times higher mutation frequencies compared with the CT library (0.12%).Importantly,the extensive diversity found in the cavity-lining residues of the CT library was maintained in both error-prone evolved libraries.Binding characteristics of the selected populationsWe used different selection strategies of various stringencies to select clones with increased testosterone binding activity. Differences were related to the amount of antigenH.Persson et al.488 at Huazhong Agricultural University on January 26, Downloaded from(testosterone-biotin)used,whether or not soluble antigen (testosterone)was present during the phage capturing step and choice of elution method.The different approaches are summarized in Table I.In order to follow the contributions of the different selec-tion parameters,the initial libraries(CT,CTep1and CTep2) and the various pools obtained after each round of selection were analyzed for binding specificity using phage ELISA. All populations were highly specific for testosterone-biotin (data not shown),suggesting that the selection procedures preserved the original specificity of the starting material. None of the populations showed reactivity tofluorescein,the specificity of clone FITC8,which was used as diversity-carrying scaffold when constructing the cavity library,nor to estradiol,a steroid highly similar to testosterone.A competitive ELISA analysis to assess the apparent affi-nities to testosterone-biotin and to soluble testosterone was also performed(Table II).In thefirst selection round,a rela-tively low selection pressure was applied,allowing the removal of non-functional clones formed by the mutagenesis process and also allowing the amplification of rare and poorly expressed scFv.Consequently,this round did not alter the obtained IC50values.In the two following rounds the selection pressure was increased considerably.As one might expect,there was a correlation between binding affinity for the biotinylated target antigen and the number of selections, with the best result obtained for strategy A H C H E L(IC50¼4Â1028M)of the third round(Table II).Thus,resulting in at least a500-fold increase in apparent affinity for testosterone-biotin compared with the starting material.The most prominent increase in binding strength was obtained by decreasing the antigen concentration.The presence of soluble testosterone(C H)during the catching step also seemed to promote lower IC50values;however,no obvious positive effect of the elution method could be observed. Whereas the affinity for testosterone-biotin showed a peak after the third selection round,it is apparent that the binding to the soluble hapten did not follow the same trend.An initial decrease in IC50values was seen when going from the first to the second selection round.This was especially appar-ent for population C2L.The subsequent selection of these clones,including higher stringency with respect to both antigen concentration and elution method,did not signifi-cantly change the affinity of the bulk population for testoster-one.In contrast,as illustrated by the differences in IC50 values of the two populations C2H and C2L(33-fold),the presence of soluble testosterone during the phage-catching phase had a major deleterious effect on the binding to free testosterone of the recovered population.This effect was further observed in selection round three,where half of the populations no longer had a detectable testosterone reactivity, suggesting that the addition of soluble testosterone comple-tely drained the populations of testosterone binders. Nevertheless,C2L of the second round of selection and four of the eight populations of the third round showed an $100-fold increase in apparent affinity for the native antigen compared with the starting populations.Screening of individual clonesA total of1152clones were picked from each of the total10 strategies from selection round two and three and their scFv were produced in soluble form.In a high-throughput ELISA the clones were assessed for binding to testosterone-biotin.A primary screening assay identified numerous clones that gave signals10–50-fold higher than the control clones TC-5and TC-20,which had previously been isolated from the CTT able II.Characteristics of the initial libraries and the different selected populationsPopulation Competitive phage ELISA onpopulations of binders High-throughput ELISA screening of individualsoluble scFvNumber ofunique scFv cNumber of scFvbinding to thesoluble hapten dIC50(TB,nM)IC50(T,nM)Primary screening a(%)Secondary screening b(%)Unselected CT.2000050000--CTep1.2000030000––CTep2.2000070000––Selection1CT*.2000050000––CTep1*.2000070000––CTep2*.2000040000––Selection2C2L100030040C2H10001000016Selection3A H C L E L50060033923/121/3A H C L E H20060018943/121/3A L C L E L70040056315/123/5A L C L E H70040028465/123/5A H C H E L40.7000033981/120/1A H C H E H200.70000441002/120/2A L C H E L2007000041923/121/3A L C H E H200.7000033903/120/3TB,testosterone-biotin.T,testosterone.a Percentage of clones having a signal greater than5000.1152clones from each strategy were analyzed in this assay.In this assay,the control antibody fragments TC-5and TC-20,which had previously been isolated from the CT library,had signals in the range of500to700.b Percentage of clones having a signal greater than10000.48clones from each strategy were analyzed in this assay.TC-5and TC-20did not result in any detectable signal in this assay.c The number of unique scFv was determined by sequencing of positive scFv as defined by the secondary ELISA screening.d The number of scFv binding the soluble hapten was assessed by competitive ELISA.In vitro evolution of an antibody fragment489 at Huazhong Agricultural University on January 26, Downloaded from。
