Expression and biological characterization of anti-CD63 single-chain variable fragment antibody in <i>Pichia pastoris</i>.

Xuedan CHEN; Zhijian CAI

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Expression and biological characterization of anti-CD63 single-chain variable fragment antibody in Pichia pastoris.

Author: Xuedan CHEN ¹ ; Zhijian CAI ¹
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1. Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310009, Zhejiang, China.
Publication Type:Journal Article
Keywords: Pichia pastoris; affinity; anti-CD63 single-chain variable fragment; secretory expression; specificity
MeSH: Single-Chain Antibodies/immunology*; Humans; Tetraspanin 30/immunology*; Recombinant Proteins/immunology*; Pichia/genetics*; Saccharomycetales/metabolism*
From: Chinese Journal of Biotechnology 2025;41(4):1440-1454
CountryChina
Language:Chinese
Abstract: To prepare antibodies that specifically recognize the conserved domain in the large extracellular loop of the CD63 protein, we expressed anti-CD63 single-chain variable fragment (scFv) antibody in Pichia pastoris in a secreted form. The purified expression product was found to bind specifically with CD63 protein and recognize CD63 on the surface of SK-MEL-28 cells. The variable region of the anti-CD63 monoclonal antibody in an anti-CD63-positive cell line was sequenced. The anti-CD63 scFv consisted of a variable heavy chain and a variable light chain linked by a flexible peptide was then designed. After codon optimization, the gene was synthesized and cloned into the expression plasmid pPICZα-A. The SacI-linearized plasmid was electroporated into P. pastoris X33, and 1% methanol were used to induce the expression of scFv. The fermentation supernatant was purified by Ni column. Anti-CD63 scFv was identified by SDS-PAGE and Western blotting, and its biological activities were analyzed by immunoblotting, immunofluorescence, cell-based ELISA, and flow cytometry. A P. pastoris strain capable of expressing and secreting anti-CD63 scFv was successfully obtained. The antibody had a molecular weight of approximately 30 kDa and specifically recognized CD63 protein. The expression of anti-CD63 scFv in P. pastoris paves the way for the production of anti-CD63 antibodies on a large-scale, which is undoubtedly an economical and effective way of antibody acquisition.