Abstract
In Escherichia coli, CGG is a rare codon occurring at a frequency of 0.57% in all mRNA. Past experiences suggest that codons occurring at this frequency create translational problems when present in high numbers or in tandem in recombinant mRNA. The coding region for a N-terminal polyhistidine tagged Herpes Simplex Virus 2 (HSV2) protease contains 11 arginine CGG codons (3 tandem CGG codons occur near the C terminus of the protein) and should generate a protein with a predicted molecular weight of 28146 daltons. However, when we expressed HSV2 protease in E. coli, the majority of product had an apparent molecular mass of >30000 daltons on SDS-PAGE gels. The aberrant protein was affinity purified by Ni-NTA chromatography, and subdigested using CNBr and trypsin. These released fragments were isolated and analyzed using ESI-TOF LC-mass spectrometry and automated N-terminal sequence analysis. We found two major species comprising a +1 frameshift at the second CGG codon in the cluster and one at the third CGG of the cluster. Translation proceeded in this new frame to the next termination codon. Coexpression of the arg3 gene, which encodes the cognate tRNA for CGG codons, enhanced production of coded HSV2 protease with a mass of 28146 daltons by 3.5–7 fold. The level of full-length authentic HSV2 protease was dependent on the amount of cognate tRNA expressed. We conclude that the ribosomes paused at the second or third CGG codon of the cluster because the level of endogenous arginyl-tRNAcGG was low. The paused ribosomes underwent a +1 frameshift at either CGG codon of the cluster. When the level of arginyl-tRNAcGG was increased, the majority of the protease had the expected molecular mass.
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References
Bogosian, G., Violand, B.N., Dorvvard-King, E.J., Workman, W.E., Jung, P.E., and Kane, J.F. Biosynthesis and incorporation into protein of norleucine by Escherichia coli. J. Biol. Chem. 264 (1989): 531–539.
Bogosian, G., Violand, B.N., Jung, P.E., and Kane, J.F. The effect of protein overexpression on mistranslation in Escherichia coli. In: W.E. Hill, A. Dahlberg, R.A. Garrett, P.B. Moore, D. Schlessinger, and J.R. Warner (eds). The Ribosome: Structure, Function, and Evolution. American Society for Microbiology, Washington/D.C. pp. 546–558, 1990.
Del Tito, B.J. Jr., Ward, J.M., Hodgson, J., Gershater, C.J.L., Edwards, H., Wysocki, L.A., Watson, F.A., Sathe, G., and Kane, J.F. Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli. J. Bacteriol. 177(1995): 7086–7091.
Dillon, P.J. and Rosen, C.A. A rapid mediod for the construction of synthetic genes using the polymerase chain reaction. Biotechniques 9 (1990): 298–300.
Kane, J.F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotech. 6 (1995): 494–500.
Kane, J.F., Balaban, S.M., and Bogosian, G. Commercial production of bovine somatotropin in Escherichia coli. In: C.S. Sikes and A.P. Wheller (eds). Surface Reactive Peptides and Polymers: Discovery and Commercialization. American Chemical Society, Washington/D.C. pp. 186–200, 1989.
Kane, J.F., Violand, B.N., Curran, D., Staten, N.R., Duffin, K.L., and Bogosian, G. Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli. Nucl. Acids Res. 20 (1992): 6707–6712.
Lu, H.S, Fausset, P.R., Sotos, L.S., Clogston, C.L., Rohde, M.F., Stoney, K.S. and Herman, A.C. Isolation and characterization of three recombinant human granulocyte colony stimulating factor His—Gin isoforms produced in Escherichia coli. Prot. Express, and Parif. 4 (1993): 465–472.
Seetharam, R., Heeren, R.A., Wong, E.Y., Braford, S.R., Klein, B., Aykent, S., Kotts, C.E., Mathis, K.J., Bishop, B.F., Jennings, M.J., Smith, C.E., and Siegel, N.R. Mistranslation in IGF-1 during over-expression of the protein in Escherichia coli using a synthetic gene containing low frequency codons. Biochem. Biophys. Res. Commun. 155 (1988): 518–523.
Spanjaard, R.A. and van Duin, J. Translation of the sequence AGG-AGG yields 50% ribosomal frameshift. Proa Natl. Acad. Sci. USA 85 (1988): 7967–7971.
Violand, B.N., Schüttler, M.R., Lawson, C.Q., Kane, J.F., Siegel, NR., Smith, C.E., and Duffin, K.L. Isolation of Escherichia coli synthesized proteins that contain s-N-acetyllysine. In: J.W. Crabb (ed.). Techniques in Protein Chemistry VI, Academic Press, New York/NY. pp. 99–106, 1995.
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McNulty, D.E. et al. (2001). Translational Problems Associated with the Rare Arginine Codon CGG in Escherichia Coli . In: Merten, OW., et al. Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology. Springer, Dordrecht. https://doi.org/10.1007/978-94-015-9749-4_12
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DOI: https://doi.org/10.1007/978-94-015-9749-4_12
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