Modification of the carboxy-terminal amino acid sequence alters the Escherichia coli expression of a gene encoding multiple repeats of a bovine growth hormone releasing factor analog.
Since investigations into the determinants of intracellular protein degradation have shown that the carboxy terminal sequence can be a critical factor for protein expression in Escherichia coli, we attempted to increase the expression of a protein containing multiple repeats of a bovine growth hormone releasing factor analog (bGRF30) by modifying the carboxy terminus with the addition of short amino acid extensions derived from stable E. coli proteins. Extensions capable of increasing bGRF30 per liter titers up to four-fold, as well as extensions that completely abolished bGRF30 expression were identified. Select C-terminal extensions were investigated further to determine the mechanism by which they affected bGRF30 expression. Analysis of mRNA levels and protein production titers suggests that extensions which increase bGRF30 titers primarily affect protein stability and ribosomal release. Negative extensions exert their influence through a more complex mechanism, appearing to interfere with the ability of ribosomes to be efficiently released from their cognate mRNA.
Trepod
C M
CM
Biology I, 7263-209-713, Pharmacia, Kalamazoo, MI 49007, USA. catherine.m.trepod@am.pnu.com
Mott
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D018628:Gene Dosage; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics*; D006207:Half-Life; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D010454:Peptide Termination Factors / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D014158:Transcription, Genetic
D010446:Peptide Fragments; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013007:Growth Hormone-Releasing Hormone; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases