Abstract
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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Andersson SGE, Kurland CG (1990) Codon preferences in free-living microorganisms. Microbiol Rev 54:198–210
Boer HA de, Hui A, Comstock LJ, Wong E, Vasser M (1983) Portable Shine-Dalgarno regions: a system for a systematic study of defined alterations of nucleotide sequences within E. coli ribosome binding sites. DNA 2:231–235
Dalbøge H, Carlsen S, Jensen EB, Christensen T, Dahl HM (1988) Expression of recombinant growth hormone in Escherichia coli: effect of the region between the Shine-Dalgarno sequence and ATG initiation codon. DNA 7:399–405
Ghrayeb J, Kimura H, Takahara M, Hsiung H, Masui Y, Inouye M (1984) Secretion cloning vectors in Escherichia coli. EMBO J 3:2437–2442
Gold L (1988) Post-transcriptional regulatory mechanisms in E. coli. Annu Rev Biochem 57:199–233
Grantham R, Grutier C, Gouy M (1980) Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome typing. Nucleic Acids Res 8:1893–1912
Hoekema A, Kastelein RA, Vasser M, Boer HA de (1987) Codon replacement in the PGKI gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression. Mol Cell Biol 7:2914–2924
Ikemura T (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes. J Mol Biol 151:389–409
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lee N, Zhang S-2, Cozzitorto J, Yang J-S, Testa D (1987) Modification of mRNA secondary structure and alteration of the expression of human interferon α1 in Escherichia coli. Gene 58:77–86
Morelle G, Mayer H (1988) Increased synthesis of human parathyroid hormone in Escherichia coli through alterations of the 5′ untranslated region. Biochem Biophys Acta 950:459–462
Olins PO, Rangwala SH (1989) A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem 264:16973–16976
O'Mahony DJ, Wang H, McConnell DJ, Jia F, Xia L, Qi S (1989) Polymorphism in porcine somatotropin cDNA sequences. Anim Genet 20:313–316
O'Mahony DJ, Wang H, McConnell DJ, Jia F, Zhou SW, Yin D, Qi S (1990) The effect of phage T7 lysozyme on the production of biologically active porcine somatotropin in Escherichia coli from a gene transcribed by T7 RNA polymerase. Gene 91:275–279
Rosenberg AH, Lade BN, Chui D, Lin S, Dunn JJ, Studier FW (1987) Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125–135
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Schauder B, McCarthy JEG (1989) The role of bases upstream of the Shine-Dalgarno region and in the coding sequences in the control of gene expression in Escherichia coli: translational and stability of mRNAs in vivo. Gene 78:59–72
Schauder B, Blöcker H, Frank R, McCarthy JEG (1987) Inducible expression of vectors incorporating the Escherichia coli atpE translational initiation region. Gene 52:279–283
Sharp PM, Li W-M (1986) Codon usage in regulatory genes in Escherichia coli does not reflect selection for ‘rare’ codons. Nucleic Acids Res 14:7737–7749
Shatzman A, Rosenberg M (1987) Expression, identification and characterization of recombinant gene products in Escherichia coli. Methods Enzymol 152:661–673
Stanssens P, Remaut E, Friers W (1985) Alterations upstream from the Shine-Dalgarno region and their effect on bacterial gene expression. Gene 36:211–223
Tessier LM, Sondermeyer P, Faure T, Dreyer D, Benevente A, Villerual D, Courtney M, Lecorq J-P (1984) The influence of mRNA primary and secondary structure on human IFN-γ gene expression in E. coli. Nucleic Acids Res 12:7663–7675
Vernet T, Tessier DC, Laliberté F, Dignard D, Thomas DY (1989) The expression in Escherichia coli of a synthetic gene coding for the precursor of papain is prevented by its own putative signal sequence. Gene 77:229–236
Vize PD, Wells JRE (1987) Spacer alterations which increase the expression of porcing growth hormone in E. coli. FEBS Lett 213:155–158
Wang H, McConnell DJ, O'Mahony DJ (1990) An efficient temperature-inducible vector incorporating the T7 gene 10 translational initiation leader region. Nucleic Acids Res 18:1070
Wood HE, Dawson MT, Devine KM, McConnell DJ (1990) Characterization of PBSX, a defective prophage of Bacillus subtilis. J Bacteriol 172:2667–2674
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wang, H., O'Mahony, D.J., McConnell, D.J. et al. Optimization of the synthesis of porcine somatotropin in Escherichia coli . Appl Microbiol Biotechnol 39, 324–328 (1993). https://doi.org/10.1007/BF00192086
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00192086