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 four-step purification and it has been demonstrated that ASNase-scFv fusion exists 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 capacity with the anti-ASNase serum decreased by comparison to the native ASNase. Moreover, here the comparison of the basic physical and chemical properties of the ASNase-scFv fusion, scFv-ASNase fusion, and native ASNase is presented. Based on the structural evidence and the biochemical analysis described in this paper, the protection mechanism proposed in our previous study was further supported. The scFv moiety of the fusion protein may confer the ASNase moiety resistance to proteolysis as a result of both steric hindrance such as blocking the cleavage sites of trypsin and a change in the electrostatic potential surface of the enzyme.
Guo
L
L
Department of Enzymology, Institute of Microbiology, Beijing, 100080, People's Republic of China.
Wang
J
J
Yan
X
X
Chen
R
R
Qian
S
S
Meng
G
G
D000818:Animals; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D001215:Asparaginase