Efficient expression of unfused human alpha D-interferon in Escherichia coli using overlapping termination and initiation codons (TGATG) in its signal sequence.
A plasmid carrying the lambda PL promoter was constructed to express efficiently unfused human alpha D-interferon (HuIFN-alpha D) in Escherichia coli using a TGATG site in its signal sequence, which occurs also in the lambda DNA sequence. The unfused nature of HuIFN-alpha D expressed by pBV867 in E. coli (BMH 71-18) was confirmed by the following evidence: first, the purified IFN showed a single band of 19.5K in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); a 27K band, representing lambda N-HuIFN-alpha D fusion protein, was not detected. Second, the peak of IFN activity coincided with the 19.5K protein. Third, the peak of absorbent material for human leukocyte IFN antibody coincided with that of IFN activity. Finally, amino-terminal sequencing of purified IFN demonstrated an unfused HuIFN-alpha D. This suggests that E. coli is able to process the signal sequence of HuIFN-alpha D. Studies on the mechanism of "translational coupling" initiation of gene expression were carried out by the construction of two hybrid plasmids and titration of the IFN activities produced by them. The level of expression by the ATG-TGATG initiation mode was found to be six times higher than that of the single ATG mode.
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007370:Interferon Type I / Q000096:biosynthesis*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D003062:Codon; D007370:Interferon Type I; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins