Nucleic Acids Research, 1989, Vol. 17, No. 8 3179-3197
© 1989
MOLECULAR BIOLOGY |
Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E.coli
Molecular Biology Research, The Upjohn Company Kalamazoo, MI 49007, USA 1Biopolymer Chemistry The Upjohn Company, Kalamazoo, MI 49007, USA
*To whom correspondence should be addressed
Received December 1, 1988. Revised March 13, 1989. Accepted March 13, 1989.
We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E. coil. The unmodified cDNA coding for mature bovine somatotropin does not produce significant amounts of BSt in E. coli using a pBR322-derived vector. However, a translation fusion with 16 codons from trpLE in front of BSt cDNA results in greater than 20% of total cell protein as the fusion product. Analysis of transcription by measuring the rate and integrity of the mRNA confirms that a post-transcriptional event is responsible for the poor expression of the BSt cDNA. There are two potential stem-loop structures in the 5' region of the mRNA which may interfere with translation. To study their effect on translation, lacZ fusions and oligonucleotide mutagenesis were carried out. The results demonstrate that the secondary structure involving the initiation codon blocks translation initiation. Removal of this stem-loop results in a 100-fold increase in BSt expression. However, the expression level is still low, amounting to only 0.5–1% of total cell pcotein. High level expression can be obtained by replacement of the beginning sequence of BSt cDNA with trpLE codons. These results suggest that in addition to the secondary structure, the nucleotide sequence or amino acid context within the beginning of BSt is incompatible with one of the steps in translation initiation.