Optimization of the synthesis of porcine somatotropin in Escherichia coli.
We report on the influence of choice of promoter and RNA polymerase, 5'-untranslated regions and ribosome binding sites, codon usage, leader peptide coding sequences and poly A tail in the 3'-untranslated region on the synthesis of porcine somatotropin (PST) in Escherichia coli. A total of 12 different constructs were tested in this study for the production of porcine somatotropin (PST) in E. coli. Several factors have significant effects on PST synthesis. In the presence of a strong promoter and a strong ribosome binding site, the next most important factor seems to be the combination of sequences at the 5'-end of the mRNA including both the 5'-untranslated region and the start of the coding sequence. Codon usage in the 5'-coding sequence per se is not important in determining the level of PST synthesis where high level expression is achieved from a strong ribosome binding site. However, where low level synthesis of recombinant PST (rPST) is achieved, codon usage in the 5'-coding sequence is important in determining the level of PST synthesis. Leader sequences dramatically reduce the level of PST synthesis. The presence of a poly A tail in the 3'-untranslated region has no significant effect on PST synthesis.
Wang
H
H
Department of Genetics, Trinity College, Dublin, Ireland.
O'Mahony
D J
DJ
McConnell
D J
DJ
Qi
S Z
SZ
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013552:Swine
D003062:Codon; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D013006:Growth Hormone