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Construction and structural modeling of a single‐chain Fv–asparaginase fusion protein resistant to proteolysis
Author(s) -
Guo Li,
Wang Jinhua,
Qian Shijun,
Yan Xiyun,
Chen Runsheng,
Meng Guangzhen
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/1097-0290(20001120)70:4<456::aid-bit11>3.0.co;2-e
Subject(s) - fusion protein , biochemistry , chemistry , protein engineering , enzyme , recombinant dna , escherichia coli , microbiology and biotechnology , peptide , proteolysis , trypsin , biology , gene
In this study, we construct a fusion protein composed of L ‐asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single‐chain Fv (scFv), which was selected from a phage‐display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N‐terminus of the enzyme moiety via a linker peptide, (Gly 4 Ser) 6 . Recombinant plasmid pET‐SLA was constructed to express scFv–ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K m value similar to that of the native enzyme for the substrate L ‐asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, α‐chymotrypsin, and rennet, at 37°C for 30 min, scFv–ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv–ASNase fusion had a much longer in vitro half‐life (9 h) in serum than the native enzyme (2 h). The three‐dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the I NSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 456–463, 2000.