Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression and Purification of a Functional Recombinant Aspartate Aminotransferase (AST) from Escherichia coli

  • Zou, Lihui (Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, Ministry of Health) ;
  • Zhao, Haijian (National Center for Clinical Laboratories, Beijing Hospital, Ministry of Health) ;
  • Wang, Daguang (Department of Laboratory Medicine, Beijing Hospital, Ministry of Health) ;
  • Wang, Meng (Department of Laboratory Medicine, Beijing Hospital, Ministry of Health) ;
  • Zhang, Chuanbao (National Center for Clinical Laboratories, Beijing Hospital, Ministry of Health) ;
  • Xiao, Fei (Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, Ministry of Health)
  • Received : 2014.02.12
  • Accepted : 2014.04.09
  • Published : 2014.07.28
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Abstract

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 $-20^{\circ}C$. 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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References

  1. Burgess-Brown NA, Sharma S, Sobott F, Loenarz C, Oppermann U, Gileadi O. 2008. Codon optimization can improve expression of human genes in Escherichia coli: a multi-gene study. Protein Expr. Purif. 59: 94-102. https://doi.org/10.1016/j.pep.2008.01.008
  2. Campos-Cavieres M, Munn EA. 1973. Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver. Biochem. J. 135: 683-693. https://doi.org/10.1042/bj1350683
  3. Chambers RS, Johnston SA. 2003. High-level generation of polyclonal antibodies by genetic immunization. Nat. Biotechnol. 21: 1088-1092. https://doi.org/10.1038/nbt858
  4. Conry-Cantilena C, VanRaden M, Gibble J, Melpolder J, Shakil AO, Viladomiu L, et al. 1996. Routes of infection, viremia, and liver disease in blood donors found to have hepatitis C virus infection. N. Engl. J. Med. 334: 1691-1696. https://doi.org/10.1056/NEJM199606273342602
  5. Cuhadar S, Koseoglu M, Atay A, Dirican A. 2013. The effect of storage time and freeze-thaw cycles on the stability of serum samples. Biochem. Med (Zagreb) 23: 70-77.
  6. Gelfand DH, Steinberg RA. 1977. Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases. J. Bacteriol. 130: 429-440.
  7. Jones S, Blackmore DJ. 1982. Observations on the isoenzymes of aspartate aminotransferase in equine tissues and serum. Equine Vet. J. 14: 311-316. https://doi.org/10.1111/j.2042-3306.1982.tb02439.x
  8. Kirsch JF, Eichele G, Ford GC, Vincent MG, Jansonius JN, Gehring H, Christen P. 1984. Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure. J. Mol. Biol. 174: 497-525. https://doi.org/10.1016/0022-2836(84)90333-4
  9. Kochkina VM. 2004. Isolation, purification, and crystallization of aspartate aminotransferase from wheat grain. Biochemistry (Mosc.) 69: 897-900. https://doi.org/10.1023/B:BIRY.0000040222.03147.01
  10. Kondo K, Wakabayashi S, Kagamiyama H. 1987. Structural studies on aspartate aminotransferase from Escherichia coli. Covalent structure. J. Biol. Chem. 262: 8648-8657.
  11. Lain-Guelbenzu B, Munoz-Blanco J, Cardenas J. 1990. Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii. Eur. J. Biochem. 188: 529-533. https://doi.org/10.1111/j.1432-1033.1990.tb15432.x
  12. Nalpas B, Vassault A, Poupon RE, Pol S, Berthelot P. 1991. An overview of serum mitochondrial aspartate aminotransferase (mAST) activity as a marker of chronic alcohol abuse. Alcohol Alcohol. Suppl. 1: 455-457.
  13. Nilsson J, Stahl S, Lundeberg J, Uhlen M, Nygren PA. 1997. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 11: 1-16. https://doi.org/10.1006/prep.1997.0767
  14. Rej R. 1978. Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues. Clin. Chem. 24: 1971- 1979.
  15. Schumann G, Bonora R, Ceriotti F, Ferard G, Ferrero CA, Franck PF, et al. 2002. 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. Clin. Chem. Lab. Med. 40: 725-733.
  16. Toussaint B, Emons H, Schimmel HG, Bossert-Reuther S, Canalias F, Ceriotti F, et al. 2010. Traceability of values for catalytic activity concentration of enzymes: a certified reference material for aspartate transaminase. Clin. Chem. Lab. Med. 48: 795-803.
  17. Turano FJ, Wilson BJ, Matthews BF. 1990. Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures. Plant Physiol. 92: 587-594. https://doi.org/10.1104/pp.92.3.587
  18. Vesper HW, Miller WG, Myers GL. 2007. Reference materials and commutability. Clin. Biochem. 28: 139-147.
  19. Yagi T, Kagamiyama H, Nozaki M. 1982. Aspartate: 2- oxoglutarate aminotransferase from Bakers' yeast: crystallization and characterization. J. Biochem. 92: 35-43. https://doi.org/10.1093/oxfordjournals.jbchem.a133929

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