EXPRESSION IN ESCHERICHIA-COLI OF THE ELONGATION-FACTOR 1-BETA GENE AND ITS NUCLEOTIDE T160C MUTANT FROM THE ARCHAEON SULFOLOBUS-SOLFATARICUS

The guanine nucleotide exchange factor EF-1 beta gene from the thermoa cidophilic archaeon Sulfolobus solfataricus (SsEF-1 beta) was amplifie d by PCR and cloned into the pT7-7 expression vector, One of four sele cted clones harbored the T160C nucleotide substitution leading to the Y54H amino acid change in a hydrophobic region of SsEF-1 beta, caused by a nucleotide misincorporation of the Tag DNA polymerase during PCR. The resulting plasmids were used to transform the Escherichia coli BL 21(DE3)pLysE strain. Upon induction with isopropyl beta-D-thiogalactop yranoside about 1.4 mg of the recombinant SsEF-1 beta (recSsEF-1 beta) and Y54HSsEF-1 beta were obtained from 1 liter of cell culture, recSs EF-1 beta and Y54HSsEF-1 beta were both able to catalyze the GDP/GTP e xchange on SsEF-1 alpha as observed with the wild-type SsEF-1 beta, In addition, the heat inactivation profiles of recSsEF-1 beta and Y54HSs EF-1 beta were identical, being both half inactivated after 30 min tre atment at 105 degrees C. These results suggest that Tyr 54 is not esse ntial for the nucleotide exchange activity and is not involved in the thermostability of SsEF-1 beta. (C) 1998 Academic Press.