Expression and purification of a functional recombinant aspartate aminotransferase (AST) from Escherichia coli.
Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and α-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 -20ºC. 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.
Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, Ministry of Health, Beijing 100730, P. R. China.
Lihui
L
Zou
Haijian
H
Zhao
Daguang
D
Wang
Meng
M
Wang
Chuanbao
C
Zhang
Fei
F
Xiao
D024442:Aspartate Aminotransferase, Mitochondrial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003062:Codon / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D003062:Codon; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D024442:Aspartate Aminotransferase, Mitochondrial