期刊
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
卷 24, 期 7, 页码 998-1003出版社
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
DOI: 10.4014/jmb.1402.02018
关键词
Aspartate aminotransferase; enzyme activity; gene recombination; protein expression and purification; quality control
资金
- National Science Foundation of China [81071431]
- National High Technology Research and Development Program [2012AA02A511]
Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and alpha-ketoglutarate to oxaloacetate and glutamate. It plays a key role in amino acid metabolism and has been widely recommended as a biomarker of liver and heart damage. Our study aimed to evaluate the extensive preparation of AST and its application in quality control in clinical laboratories. We describe a scheme to express and purify the 6His-AST fusion protein. An optimized sequence coding AST was synthesized and transformed into Escherichia coli BL21 (DE3) strain for protein expression. Ideally, the fusion protein has a volumetric productivity achieving 900 mg/l cultures. After affinity chromatography, the enzyme activity of purified AST reached 150,000 U/L. Commutability assessment between the engineered AST and standard AST from Roche suggested that the engineered AST was the better candidate for the reference material. Moreover, the AST showed high stability during long-term storage at -20 degrees C. In conclusion, the highly soluble 6His-tagged AST can become a convenient tool for supplying a much better and cheaper standard or reference material for the clinical laboratory.
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