title: Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis.
abstract: 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, alpha-chymotrypsin, and rennet, at 37 degrees 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.
authors.affiliation: Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR. China.
authors.forename: L
authors.initials: L
authors.lastname: Guo
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: S
authors.initials: S
authors.lastname: Qian
authors.forename: X
authors.initials: X
authors.lastname: Yan
authors.forename: R
authors.initials: R
authors.lastname: Chen
authors.forename: G
authors.initials: G
authors.lastname: Meng
mesh_terms: D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015723:Gene Library; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D001215:Asparaginase