TY - JOUR
T1 - Construction and structural modeling of a single-chain Fv-asparaginese fusion protein resistant to proteolysis
AU - Guo, Li
AU - Wang, Jinhua
AU - Qian, Shijun
AU - Yan, Xiyun
AU - Chen, Runsheng
AU - Meng, Guangzhen
PY - 2000/11/20
Y1 - 2000/11/20
N2 - 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, (Gly4Ser)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 INSIGHT 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. (C) 2000 John Wiley and Sons, Inc.
AB - 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, (Gly4Ser)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 INSIGHT 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. (C) 2000 John Wiley and Sons, Inc.
KW - Bacterial expression
KW - Fusion protein
KW - L-asparaginase
KW - Recombinant DNA technology
KW - Resistance to proteolysis
KW - Single-chain Fv (scFv)
KW - Three-dimensional structure modeling
UR - http://www.scopus.com/inward/record.url?scp=0034694204&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034694204&partnerID=8YFLogxK
U2 - 10.1002/1097-0290(20001120)70:4<456::AID-BIT11>3.0.CO;2-E
DO - 10.1002/1097-0290(20001120)70:4<456::AID-BIT11>3.0.CO;2-E
M3 - Article
C2 - 11005928
AN - SCOPUS:0034694204
VL - 70
SP - 456
EP - 463
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 4
ER -