Isocitrate dehydrogenase from Thermus aquaticus YT1: purification of the enzyme and cloning, sequencing, and expression of the gene.

Applied and Environmental Microbiology
K Miyazaki

Abstract

Isocitrate dehydrogenase from an extremely thermophilic bacterium, Thermus aquaticus YT1, was purified to homogeneity, and the gene was cloned by using a degenerate oligonucleotide probe based on the N-terminal sequence. The gene consisted of a single open reading frame of 1,278 bp preceded by a Shine-Dalgarno ribosome binding site, and a terminator-like sequence was detected downstream of the open reading frame. The G+C content of the coding region was 65%, and that of the third nucleotide of the codons was 93%. The amino acid sequence of the enzyme showed a relatively low level of similarity to the counterpart from T. thermophilus (35% identity) but showed higher levels of similarity (54 to 69% identity) to the other bacterial counterparts so far reported, including those from Escherichia coli, Bacillus subtilis, Vibrio sp., and Anabaena sp. The cloned gene was highly expressed in E. coli and easily purified to homogeneity by heat treatment (70 degrees C, 30 min) and DEAE column chromatography to yield approximately 10 mg of protein from 1 g of wet cells. The recombinant enzyme showed high thermostability and almost the same heat denaturation profile as the intact enzyme purified from the thermophile cells, implying that the ...Continue Reading

References

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Nov 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·J H HurleyR M Stroud
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Sep 14, 1993·Biochemistry·A M Dean, D E Koshland

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Citations

Nov 15, 2005·The Protein Journal·Adoración Rodríguez-ArnedoMaría-José Bonete
Mar 24, 2000·Archives of Biochemistry and Biophysics·D B Karr, D W Emerich

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