Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli

Abstract

A pBR322-T. leu hybrid plasmid was constructed which contains a 3.75 Md HindIII-fragment derived from Thermus thermophilus HB27 chromosomal DNA. In the Escherichia coli host, this plasmid coded for the beta-IPM dehydrogenase (product of leuB) activity, the optimal temperature of which was 80 degrees C, suggesting that information on the thermostability of the enzyme lies in its structural gene. 10-day propagation of E. coli [pBR322-T.leu] at 37 degrees C decreased the temperature optimum from 80 degrees to 75 degrees C. This change, which was found to depend on the plasmid but not on the host cells, might be due to selection of some mutation at the non-restrictive temperature of 37 degrees C. Our results suggest that the 3.75 Md HindIII-fragment of pBR322-T.leu carries a promoter of the thermophile, which could function in E. coli.