Improved Recombinant Tandem Expression of Translation Initiation Factor IF2 in RNase E Deficient E. coli Cells

Abstract

The prokaryotic translation initiation factor IF2 exists in a varying number of nested forms in different species. In E. coli three natural forms exist, IF2α, IF2β and IF2γ differing only in the N-terminal: IF2β and IF2γ lack 158 and 165 amino acid residues, respectively, as compared to IF2α. We have earlier shown that the smaller forms of IF2 are not the result of a specific proteolysis of IF2α, but produced from individual translation initiation sites in the mRNA. However it has not been known whether the expression in E. coli of IF2β and IF2γ is dependent on or related to a posttranscriptional processing of the polycistronic nusA operon, containing infB, the gene for IF2. Here we have used S1 mapping to study the existence of such mRNA processing in the region between the initiation sites for IF2α and IF2β/IF2γ. The results show a Ribonuclease E cleavage site at position +200 in the infB mRNA between the translation initiation sites. However, studies of the overexpression of the different forms of IF2 show that the relative expression of IF2α and IF2β/IF2γ is independent of RNase E activity. Thus E. coli exhibits a true tandem translation of intact infB mRNA with multiple in-frame translation initiation sites resulting in gene products of different sizes. An additional observation is a significant increase in the level of overexpression of IF2 in cells devoid of RNase E activity. We conclude that due to lack of RNase E activity, the amount of plasmid-transcribed infB mRNA available for translation is accumulated, resulting in an elevated amount of recombinant IF2. This observation may have a more general application within the field of recombinant protein production and expression efficiency.