Development of a compact anti-BAFF antibody in Escherichia coli.
Recombinant antibodies, especially single-chain antibody fragment (scFv), can be applied as detection reagents and even substitute for some reagents used in immunoassays. For scFv fragments, there is no such universal system available up to now. We have constructed vectors for the convenient, rapid expression of a novel compact antibody composed of anti-B-cell-activating factor of the TNF family (BAFF) scFv and the Fc portion (the hinge region, CH2, and CH3 domains) of the human IgG1 in Escherichia coli. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The scFv-Fc antibody was demonstrated to retain high binding affinity to antigen, including membrane-bound BAFF and soluble BAFF, and to possess some human IgG crystallizable fragment domain functions, such as human complement C1q and protein A binding. Both size-exclusion high-performance liquid chromatography column analysis and Western blotting of proteins subjected to nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested that scFv-Fc antibody is homodimeric with relative molecular mass of 110 kDa. These findings suggest that the compact antibody may be useful in diagnostic application for the prediction of BAFF relevant to autoimmune diseases in human.
Cao
Peng
P
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210097, Jiangsu, People's Republic of China.
Zhang
Shuangquan
S
Gong
Zhunan
Z
Tang
Xiangming
X
Cao
Meng
M
Hu
Yunlong
Y
D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001327:Autoimmune Diseases / Q000175:diagnosis; D053264:B-Cell Activating Factor / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D015922:Complement C1q / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism
D000906:Antibodies; D053264:B-Cell Activating Factor; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D015922:Complement C1q