Purification of Escherichia coli L-asparaginase mutants by a native polyacrylamide gel electrophoresis.
The antigenicity of L-asaparaginase (L-ASP) has been problematic for the treatment of leukemia for many years. In order to establish a relationship between the antigenic epitope of L-asparaginase and its antigenicity, several L-asparaginase mutants (mL-ASPs) are constructed and expressed. To effectively purify these enzyme mutants for further investigation, a native preparative polyacrylamide gel electrophoresis is developed. The simplicity and reproducibility of this approach permits the purification of different mutants from the crude enzyme extracts, with a sufficient activity to perform immunological and biological studies. Furthermore, the newly developed method is efficient and cost-effective compared with other methods, such as column chromatography and affinity chromatography. As a result, the enzyme mutants with specific activity of 300 approximately 400 U/mg are obtained by the single-step purification with a high degree of purity.
Wei
Yujun
Y
The School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, People's Republic of China.
Chen
Jianhua
J
Jia
Ruibo
R
Wang
Min
M
Wu
Wutong
W
D001215:Asparaginase / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D001215:Asparaginase