Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis

In this study, we construct a fusion protein composed of L-asparaginase (AS Nase; from Escherichia coil AS 1.357) and a protective single-chain Fv (scF v), 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-termi nus 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 level s in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The sol uble fusion protein exhibited about 82% of the enzymatic activity of the na tive 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) follow ing treatment with trypsin, a-chymotrypsin, and rennet, at 37 degreesC 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) ScF v-ASNase fusion had a much longer in vitro half-life (9 h) in serum than th e native enzyme (2 h). The three-dimensional structure of the fusion protei n was obtained by modeling with the Homology and Discover modules of the IN SIGHT II software package. On the basis of the structural evidence and bioc hemical properties, we propose that the scFv moiety of the fusion protein m ay confer ASNase moiety resistance to proteolysis as a result of both steri c hindrance and a change in the electrostatic surface of the enzyme. (C) 20 00 John Wiley & Sons, Inc.