Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin.
Four non-inhibitory specific single-chain Fv (sc Fv) fragments directed against L-asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage-display scFv library. The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase-scFv46 complex had been treated with trypsin for 30 min at 37 degrees C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression vector pET-21a and expressed at high levels (about 45% of total cell protein) in E. coli BL21 (DE3) as inclusion bodies. The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase-scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 degrees C. The results demonstrated that scFv fragments prepared by phage-antibody library technology could be used to protect target proteins.
Guo
L
L
Department of Enzymology, Institute of Microbiology, Academia Sinica, Beijing 100080, People's Republic of China.
Yan
X
X
Qian
S
S
Meng
G
G
D001215:Asparaginase / Q000235:genetics* / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D005786:Gene Expression Regulation; D015723:Gene Library; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012641:Selection, Genetic; D017421:Sequence Analysis; D012995:Solubility; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D014357:Trypsin; D001215:Asparaginase