Probing the antigenicity of E. coli L-asparaginase by mutational analysis.
A strategy, termed alanine-scanning mutagenesis, was used to identify the amino acid residues which are critical to the antigenicity of Escherichia coli L-asparaginase (L-ASP). Three continuous alkaline residues, 195RKH197, were mutated to Ala selectively. Four mutant recombinant L-ASPs were constructed and expressed in E. coli, and then purified. The purified mutants showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were more than 95% pure by reverse high-performance liquid chromatography. The activities of wild-type and mL-ASPs in the fermentative medium were all about 130 U/mL. The change from 195RKH197 to 195AAA197 reduced the antigenicity of the enzyme greatly as shown in competition enzyme-linked immunosorbent assay using polyclonal antibodies raised against the wild-type L-ASP from rabbits. The results show that residues 195RKH197 of E. coli L-ASP are critical to its antigenicity.
Jianhua
Chen
C
The School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China. jhchen@cpu.edu.cn
Yujun
Wei
W
Ruibo
Jia
J
Min
Wang
W
Wutong
Wu
W
D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology*; D001215:Asparaginase / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D002457:Cell Extracts; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
D000906:Antibodies; D000941:Antigens; D002457:Cell Extracts; D011994:Recombinant Proteins; D001215:Asparaginase