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重组基因工程抗体和蛇毒C型凝集素类似蛋白在毕赤酵母中表达及结构功能研究
【作者】 胡思怡；
【导师】 刘兢；
【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2006， 博士
【摘要】 巴斯德毕赤甲醇营养型酵母(Pichia Pastoris)属于低等的真核表达系统，与其他表达系统相比，具有遗传操作简单、蛋白折叠和二硫键加工能力较强、易于高密度发酵、蛋白表达量较高等优点，已经成为重组蛋白研究和商业化生产的重要表达系统之一。本论文讨论了抗ErbB2基因工程抗体和重组蛇毒毒素蛋白在毕赤酵母中表达，以及结构和功能关系研究中，主要包括以下三部分内容： 1．密码子优化的抗ErbB2单链抗体在Pichia中表达、纯化和活性鉴定。ErbB2／HER2／P185属于具有酪氨酸激酶的表皮生长因子受体家族，在细胞生长发育和组织分化中起到了重要作用，ErbB2过表达经常在细胞癌化和肿瘤发生中起关键的作用，因此成为肿瘤诊断和治疗的一个重要靶分子。本实验室研制了一株具有肿瘤生长抑制活性的抗ErbB2单克隆抗体mA21，并且构建和表达了工程化的单链嵌和抗体chA21。本研究就是在前期工作基础上合成了密码子优化的单链抗体scFv全基因，并且在Pichia酵母中获得了高效表达，进一步优化表达条件后单链抗体表达量达到了10mg/L。单链抗体经Ni2+亲和柱一步纯化后，即可用于晶体生长、亲和力测定等结构和功能研究。 2．抗ErbB2胞外区抗体A21的表位鉴定、晶体结构分析和抗肿瘤机理的初步研究。我们联合使用噬菌体随机肽库表面展示技术、抗原在原核系统中分段表达、抗原点突变扫描等实验精细测定了A21抗体识别的抗原表位和关键性残基。在蛋白质结构晶体实验室的帮组下解析了A21单链抗体2.1A分辨率的晶体结构。并且利用大分子对接软件，基于实验结果预测并验证了A21抗体与ErbB2抗原可能的相互作用模型。结果显示A21抗体主要识别ErbB2胞外区靠近N端I区的空间表位。我们还测定了A21抗体对肿瘤细胞株的增殖抑制活性、抗体的内吞作用、抗体对ErbB2磷酸化的影响、以及抗体诱导的ErbB2受体下调作用，进一步解释了A21抗体表位和抗体功能之间的联系。 3．重组蛇毒C型凝集素类似蛋白ACFI在Pichia酵母中表达、纯化和结构功能关系研究。蛇毒C型凝集素类似蛋白具有非常相似的二硫键连接的异二聚体结构，但是具有多种不同的生物学功能。ACFI是从皖南尖吻蝮蛇蛇毒中分离到的一种可以与凝血因子结合的具有抗凝活性的C型凝集素类似蛋白。我们设计了一种新的共转化策略，使用两个不同抗生素选择标记的载体用于筛选ACFI两个亚基共表达的重组菌株。结果显示，重组ACFI亚基在Pichia酵母中单独表达时形成同二聚体，共表达时则可以形成异二聚体。重组ACFI具有与天然蛋白类似的凝血因子结合活性和抗凝活性，但是重组亚基却没有这些功能，说明ACFI两个亚基对维持其正常的生物学功能具有非常重要和不可替代的作用。我们的结果提示Pichia系统在蛇毒C型凝集素类似蛋白结构和功能关系的研究中具有重要的应用价值。
【Abstract】 The methylotrophic yeast Pichia Pastoris is a single-cell microorganism as well as a eukaryote. The P. pastoris expression system combines some of the advantages of both the bacterial and eucaryotic systems, e.g. efficient in disulfide-bond folding and post-transcriptional processing, and also easy for manipulation, culture and fermentation, thus it has been widely used for high-level expression of thousands of heterologous proteins for both basic laboratory research and industrial manufacture. In my dissertation, we showed the application of P. pastoris in expressing engineered antibody fragments and recombinant snake toxins for structure-function study, mainly including three chapters:1. Codon optimization, expression, purification and characterization of the anti-ErbB2 single-chain antibody (scFv). ErbB2/HER2/P185 belongs to the epidermal growth factor receptor (EGFR) family, and as a tyrosine kinase receptor it plays an important role in regulating normal cell growth and differentiation. ErbB2-overexpression is often associated with cell transformation and tumorigenesis, making it an attractive target in tumor diagnoses and therapy. In the previous study, we generated a monoclonal antibody mA21 against ErbB2 extracellular domain, and the engineered single-chain chimeric antibody chA21 also exhibited potentials in inhibition of ErbB2-overexpressing tumor cells. We synthesized the condo-optimal full-length scFv gene and expressed it in P. pastoris. After further optimization of culture and induction conditions, the scFv expression level was achieved to 10mg/L. The secreted scFv was purified by one-step Ni2+ chelating chromatography and then used for biological function studies.2. Epitope mapping, crystal structure analysis and functional mechanism study of the A21 anti-ErbB2 antibodies. We precisely located the A21 epitope site and determined possible contacing residues by combinatorial utilization of phage peptide library screening, GST-fusion domain expression and mutagenesis scanning in E.coli. We then determined the 2.1 A crystal structure of the scFv expressed in P. pastoris and proposed the A21 scFv-ErbB2 complex model by molecular docking. These results suggested that A21 recognizes a conformational epitope mainly compassing ErbB2 subdomain I. We also determined the internalization ability of A21 antibodies as well as their effects on tumor cell growth, ErbB2 phosphorylation and down-regulation. Based on these findings we further discussed the relationship between the epitopes of anti-ErbB2 antibodies and functional mechanisms.3. Expression, purification and functional study of a recombinant C-type lectin-like protein (CLP) ACFI in P. pastoris. Snake CLPs have similar structures consisting of two disulfide-linked subunits but diverse functions. ACFI is an anticoagulant CLP isolated from Agkistrodon acutus venom, which could bind to coagulation factors IX and X. We expressed the two subunits of ACFI in P. pastoris by a novel co-transforming strategy using two vectors with different selectable markers. Recombinant homodimers were secreted when the ACFI subunits were expressed alone, while heterodimers (rACFI) secreted when two subunits co-expressed. rACFI retained both the coagulation factors-binding activity and APTT-prolonging activity similar to nature ACFI, but recombinant homodimers completely lost these activities, indicating the heterodimerization of two subunits is required for its function. It also suggests that P. pastoris is a promising system for structure-function studies of snake CLPs.