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Site-directed mutagenesis of aspartate aminotransferase from E. coli
Authors:B A Malcolm  J F Kirsch
Affiliation:1. School of Life Sciences, Jawaharlal Nehru University, New Delhi, Delhi 110067, India;2. Technology Bhavan, Ministry of Science and Technology, New Mehrauli Road, New Delhi 110016, India;1. College of Chemical Engineering, Fuzhou University, Fuzhou 350102, China;2. Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China;3. Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, China;4. Department of Chemistry, Engineering Center of Catalysis and Synthesis for Chiral Molecules, Fudan University, Shanghai 200433, China;5. Institute of Pharmaceutical Science and Technology, Fuzhou University, China
Abstract:The gene for aspartate aminotransferase from E. coli (aspC) was subcloned into M13 phage and sequenced using the Sanger dideoxy method with synthetic oligonucleotide primers. A mutant gene was constructed using site-directed mutagenesis techniques in which the codon for the lysine that forms the Schiffs base with pyridoxal phosphate was replaced with one coding for alanine. The mutant gene was expressed under control of the Tac promoter to overproduce a mutant protein lacking enzymatic activity.
Keywords:
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