Biased expression, under the control of single promoter, of human interferon alpha-2b and Escherichia coli methionine amino peptidase genes in E. coli, irrespective of their distance from the promoter

Human interferon alpha-2b and Escherichia coli methionine amino peptidase genes were cloned independently as well as bicistronically in expression plasmid pET-21a [+]. Production of human interferon alpha-2b was comparable to that of E. coli methionine amino peptidase when these genes were expressed independently in E. coli BL21-CodonPlus [DE3]-RIL. However, human interferon alpha-2b was produced in a much less amount whereas there was no difference in the production of methionine amino peptidase when the encoding genes were expressed bicistronically. It is important to note that human interferon alpha-2b was the first gene in order, after the promoter and E. coli methionine amino peptidase was the next with a linker sequence of 27 nucleotides between them

Truncated type II isopentenyl diphosphate isomerase from hyperthermophilic achaeon thermococcus kodakaraensis implicates the necessity of its N-terminal amino acid residues in protein thermostability

The enzyme isopentenyl diphosphate isomerase [IDI, EC 5.3.3.2] interconverts isopentenyl diphosphate and dimethylallyl diphosphate. We had previously cloned Tk-idi gene encoding the thermostable Tk-IDI enzyme from Thermococcus kodakaraensis KOD1. Four putative start codons were found on Tk-idi gene at 123, 213, 297 and 321 positions downstream of the first start codon. In the present work four mutants were obtained by deleting 123, 213, 297 and 321 nucleotides from the 5'-end of Tk-idi gene to obtain Tk-idim, Tk-idim1, Tk-idim2, and Tk-idim3, respectively. When we tried to express these truncated genes in Escherichia coli only Tk-idim was expressed in the active form. The product, Tk-IDIM, was purified and characterized. The molecular mass of the enzyme, estimated by gel filtration chromatography, was 300 kDa which indicated that the truncated enzyme retained the octameric form. The removal of 41 N-terminal amino acids did not exhibit a significant effect on the enzyme activity however, the thermostability of the enzyme decreased. The decrease in thermostability of Tk-IDIM correlated well with the results of circular dichroism [CD] analysis and structural modeling