Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E. coli.
We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E. coli. 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 protein. 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.
Tomich
C S
CS
Molecular Biology Research, Upjohn Company, Kalamazoo, MI 49007.
Olson
E R
ER
Olsen
M K
MK
Kaytes
P S
PS
Rockenbach
S K
SK
Hatzenbuhler
N T
NT
D003001:Cloning, Molecular; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D013006:Growth Hormone / Q000235:genetics*; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid
D012333:RNA, Messenger; D011994:Recombinant Proteins; D013006:Growth Hormone