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Selecting and expressing protective single‐chain Fv fragment to stabilize L‐asparaginase against inactivation by trypsin
Author(s) -
Guo Li,
Yan Xiyun,
Qian Shijun,
Meng Guangzhen
Publication year - 2000
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba19990062
Subject(s) - trypsin , escherichia coli , proteolysis , microbiology and biotechnology , recombinant dna , chemistry , inclusion bodies , phage display , enzyme , antibody , biochemistry , biology , gene , peptide , immunology
Four non‐inhibitory specific single‐chain Fv (sc Fv) fragments directed against L‐asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage‐display scFv library. The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase–scFv46 complex had been treated with trypsin for 30 min at 37 °C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression vector pET‐21a and expressed at high levels (about 45% of total cell protein) in E. coli BL21 (DE3) as inclusion bodies. The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase–scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 °C. The results demonstrated that scFv fragments prepared by phage‐antibody library technology could be used to protect target proteins.