Improved recombinant tandem expression of translation initiation factor IF2 in RNASE E deficient E. coli cells.
The prokaryotic translation initiation factor IF2 exists in a varying number of nested forms in different species. In E. coli three natural forms exist, IF2 alpha, IF2 beta and IF2 gamma differing only in the N-terminal: IF2 beta and IF2 gamma lack 158 and 165 amino acid residues, respectively, as compared to IF2 alpha. We have earlier shown that the smaller forms of IF2 are not the result of a specific proteolysis of IF2 alpha, but produced from individual translation initiation sites in the mRNA. However it has not been known whether the expression in E. coli of IF2 beta and IF2 gamma 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 IF2 alpha and IF2 beta/IF2 gamma. 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 IF2 alpha and IF2 beta/IF2 gamma 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.
Mortensen
K K
KK
Department of Chemistry, Aarhus University, Denmark.
Hajnsdorf
E
E
Regnier
P
P
Sperling-Petersen
H U
HU
D001426:Bacterial Proteins / Q000096:biosynthesis*; D001483:Base Sequence; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010448:Peptide Initiation Factors / Q000096:biosynthesis*; D010957:Plasmids; D039881:Prokaryotic Initiation Factor-2; D012333:RNA, Messenger / Q000378:metabolism
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012333:RNA, Messenger; D004722:Endoribonucleases; C022537:ribonuclease E