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Construction, Purification, and Characterization of Anti-BAFF scFv–Fc Fusion Antibody Expressed in CHO/dhfr− Cells

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Abstract

Elevated levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, we have constructed a vector for the expression of a novel compact antibody composed of anti-BAFF single-chain antibody fragment (scFv) and the Fc region (the hinge region, CH2, and CH3 domains) of human IgG1 in Chinese hamster ovary cells. The scFv–Fc fusion protein, showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges, was affinity-purified on protein A Sepharose from culture supernatant. The scFv–Fc antibody was demonstrated to retain high binding affinity to antigen and prolonged clearance time in blood and to possess some human IgG crystallizable fragment effector functions such as protein A binding and antibody-dependent cellular cytotoxicity. These results suggest that this recombinant antibody may have therapeutic applications in the therapy of autoimmune disorders mediated by BAFF.

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Abbreviations

BLyS:

B lymphocyte stimulator

BAFF:

B-cell activating factor belonging to the TNF family

BSA:

Bovine serum albumin

FBS:

Fetal bovine serum

Ab:

Antibody

TALL-1:

TNF- and ApoL-related leukocyte-expressed ligand 1

THANK:

TNF homologue that activates apoptosis, nuclear factor-kB, and c-Jun NH2 terminal kinase

TNF:

Tumor necrosis factor

Fc:

Crystallizing fragment, consists of CH2 and CH3, glycosylation site, not antigen binding, but responsible for effector functions

G418:

Neomycin

VH:

Immunoglobulin heavy-chain variable region

VL:

Immunoglobulin light-chain variable region

CH2 and CH3:

Immunoglobulin heavy-chain constant regions 2 and 3

HRP:

Horseradish peroxidase

mAb:

Monoclonal antibody

TBST:

Tris-buffered saline Tween-20

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Acknowledgments

This work was supported by grants of the Nanjing Normal University and Jiangsu Province Graduate Innovation Project (No. 1612005024) and the International Cooperation of Jiangsu Province (No. BZ2007078). We are particularly grateful to Dr Ling Wang in Nanjing Medical University for help and technical support in the experiments of Flow Cytometry Analysis.

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Authors and Affiliations

  1. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu, 210046, People’s Republic of China

    Meng Cao, Huaijiang Yan, Wuguang Lu, Fang Ren, Yunlong Hu & Shuangquan Zhang

  2. Laboratory of Molecular Medicine, Jiangsu Province Institute of Traditional Chinese Medicine, Nanjing, Jiangsu, 210028, People’s Republic of China

    Peng Cao

  3. Jiangsu Province Engineering Research Center for Biomedical Function Materials, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu, 210046, People’s Republic of China

    Shuangquan Zhang

  4. Department of Microbiology, Parasitology and Immunology, College of Basic Medical Science, Southeast University, Nanjing, Jiangsu, 210009, People’s Republic of China

    Meng Cao

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  1. Meng Cao
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Correspondence to Shuangquan Zhang.

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Cao, M., Cao, P., Yan, H. et al. Construction, Purification, and Characterization of Anti-BAFF scFv–Fc Fusion Antibody Expressed in CHO/dhfr− Cells. Appl Biochem Biotechnol 157, 562–574 (2009). https://doi.org/10.1007/s12010-008-8434-6

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