Characterization of L-asparaginase fused with a protective ScFv and the protection mechanism

A fusion protein of the protective scFv linked to the C-terminus of ASNase via (Gly(4)Ser)(6) peptide was constructed. The ASNase-scFv fusion protein expressed in Escherichia coli exists mainly in the form of inclusion bodies , and a small amount of it was soluble. The soluble form was purified by fo ur-step purification and it has been demonstrated that ASNase-scFv fusion e xists as a dimer. By assay of the stability against proteolysis, the ASNase -scFv fusion was found to be more stable than native ASNase but less stable than scFv-ASNase fusion. The results of immunological assay indicated that the immunogenicity of the fusion proteins increased while their binding ca pacity with the anti-ASNase serum decreased by comparison to the native ASN ase. Moreover, here the comparison of the basic physical and chemical prope rties of the ASNase-scFv fusion, scFv-ASNase fusion, and native ASNase is p resented. Based on the structural evidence and the biochemical analysis des cribed in this paper, the protection mechanism proposed in our previous stu dy was further supported. The scFv moiety of the fusion protein may confer the ASNase moiety resistance to proteolysis as a result of both steric hind rance such as blocking the cleavage sites of trypsin and a change in the el ectrostatic potential surface of the enzyme. (C) 2000 Academic Press.