Expression and Purification of a Functional Recombinant Aspartate Aminotransferase (AST) from Escherichia coli = Expression and Purification of a Functional Recombinant Aspartate Aminotransferase (AST) from Escherichia coli

다국어 초록 (Multilingual Abstract)

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.

참고문헌 (Reference)

1 Toussaint B, "Traceability of values for catalytic activity concentration of enzymes: a certified reference material for aspartate transaminase" 48 : 795-803, 2010

2 Cuhadar S, "The effect of storage time and freeze-thaw cycles on the stability of serum samples" 23 : 70-77, 2013

3 Kondo K, "Structural studies on aspartate aminotransferase from Escherichia coli. Covalent structure" 262 : 8648-8657, 1987

4 Conry-Cantilena C, "Routes of infection, viremia, and liver disease in blood donors found to have hepatitis C virus infection" 334 : 1691-1696, 1996

5 Vesper HW, "Reference materials and commutability" 28 : 139-147, 2007

6 Campos-Cavieres M, "Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver" 135 : 683-693, 1973

7 Lain-Guelbenzu B, "Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii" 188 : 529-533, 1990

8 Turano FJ, "Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures" 92 : 587-594, 1990

9 Jones S, "Observations on the isoenzymes of aspartate aminotransferase in equine tissues and serum" 14 : 311-316, 1982

10 Kirsch JF, "Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure" 174 : 497-525, 1984

11 Kochkina VM., "Isolation, purification, and crystallization of aspartate aminotransferase from wheat grain" 69 : 897-900, 2004

12 Schumann G, "IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase" 40 : 725-733, 2002

13 Chambers RS, "High-level generation of polyclonal antibodies by genetic immunization" 21 : 1088-1092, 2003

14 Gelfand DH, "Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases" 130 : 429-440, 1977

15 Burgess-Brown NA, "Codon optimization can improve expression of human genes in Escherichia coli: a multi-gene study" 59 : 94-102, 2008

16 Yagi T, "Aspartate: 2-oxoglutarate aminotransferase from Bakers’ yeast: crystallization and characterization" 92 : 35-43, 1982

17 Rej R., "Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues" 24 : 1971-1979, 1978

18 Nalpas B, "An overview of serum mitochondrial aspartate aminotransferase (mAST) activity as a marker of chronic alcohol abuse" 1 : 455-457, 1991

19 Nilsson J, "Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins" 11 : 1-16, 1997