Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli.
The conditions necessary for high-level expression of methionyl bovine growth hormone (Met-bGH) in Escherichia coli were investigated. Plasmids were constructed that contain a thermoinducible runaway replicon and either the E. coli tryptophan or lipoprotein promoter and ribosome binding sites, which served as transcriptional and translational initiation sites for the expression of the bGH gene. The expression of Met-bGH was low with either system. However, expression levels of up to 30% of total cell protein were obtained after the introduction of additional codons 3' to the initiating AUG codon, thus altering the NH2-terminal amino acid sequence of bGH. To obtain high-level expression of Met-bGH a two-cistron system was constructed in which the codons that enhanced the expression of bGH were incorporated into the first cistron, and the coding region for Met-bGH was incorporated into the second cistron. This approach may be generally applicable to achieving high-level expression of a gene that contains NH2-terminal sequences that do not allow for its efficient expression. Analyses of the stabilities of the bGH derivatives and their transcripts in vivo suggested that the variations in the level of expression were due to variations in the efficiency of mRNA translation.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D013006:Growth Hormone / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification
D012333:RNA, Messenger; D013006:Growth Hormone; D004262:DNA Restriction Enzymes