[Cloning and expression in E. coli of 2,5-DKG reductase I from Corynebacterium].
Two different 2,5-DKG reductases was purified from Corynebacterium sp. SCB3058, then its genomic DNA was used as template, a segment containing 2,5-DKG reductase I was amplified by PCR and cloned into pGEM3zf(+) to obtain the recombinant plasmid pGEM813. The sequence analysis showed that the cloned segment was 1107 bp in length, contained a single open reading frame of 834 nucleotides, which encoded a 34 kD protein consisted of 278 amino acids. After the primary control sequence was deleted, the expression vector pBL4 was constructed by plasmid pBL. With induction of temperature, a 34 kD protein was specifically expressed in E. coli DH5 alpha in high level. The expressed recombinant protein accounted for 20% of the total cell protein and had high specific enzyme activity. In spite of 30 degrees C, 37 degrees C or 42 degrees C induction, the specific activity of enzyme was almost the same level. These results indicate that most of the recombinant protein induced at 42 degrees C was existed with inclusion bodies. This work was the bases of constructing a recombinant Erwinia which can produce 2-KLG directly from D-glucose by one-step fermentation.
Chen
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Shanghai Research Centre of Biotechnology, Chinese Academy of Sciences, Shanghai 200233.
Yin
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D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D013401:Sugar Alcohol Dehydrogenases; C053163:2,5-diketo-D-gluconate 5-reductase