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Production and characterization of a bacterial single-chain antibody fragment specific to B-cell-activating factor of the TNF family.

Abstract：

:An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction. The construct VH-linker-VL was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli BL21(DE3) as inclusion bodies. After extraction from the E. coli cells, the inclusion bodies were solubilized and denatured in the presence of 8M urea. The expressed scFv fusion proteins were purified by Ni(2+)-IDA His-bind resin and finally renatured by dialysis. The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and enzyme-linked immunosorbent assay. The result revealed that the ABL-1 scFv retains the specific binding activity to BAFF with an affinity constant of 0.9x10(-8)molL(-1).

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Cao P,Tang XM,Guan ZB,Diao ZY,Zhang SQ

doi

10.1016/j.pep.2005.04.022

keywords:

subject

Has Abstract

pub_date

2005-10-01 00:00:00

pages

157-64

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(05)00187-7

journal_volume

43

pub_type

杂志文章

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