Recombinant Antibodies And Snake Venom C-type Lectin-like Protein Expression In Pichia Pastoris And The Structure And Function

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.