Influence of Insertion of the Last Sense Codon on Expression Efficiency of Green Fluorescent Protein Gene in Escherichia coli


We studied the relationship between insertion of the last sense codon (i.e., the codon preceding the stop codon) and the efficiency of gene expression. We inserted 64 kinds of last sense codon at the 5’ end of the stop codon of the green fluorescent protein (GFP) gene and introduced the modified GFP genes into Escherichia coli (E. coli). Measuring the fluorescence intensity of the GFP produced in E. coli showed that the last sense codon influenced GFP gene expression and when CCG was inserted as the last sense codon, fluorescence intensity of E. coli was increased to 2.09 fold. On the other hand, insertion of CUA caused decrease of fluorescence intensity to 0.33 fold. We hope that our findings, which may be applicable to gene engineering, will be useful for further studies of protein expression.

Share and Cite:

Hao, X. , Inoue, S. and Ishikawa, M. (2015) Influence of Insertion of the Last Sense Codon on Expression Efficiency of Green Fluorescent Protein Gene in Escherichia coli. Journal of Materials Science and Chemical Engineering, 3, 13-18. doi: 10.4236/msce.2015.36003.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Tolia, N.H. and Joshua-Tor, L. (2006) Strategies for Protein Coexpression in Escherichia coli. Nature Methods, 3, 55-64.
[2] Rosano, G.L. and Ceccarelli, E.A. (2014) Recombinant Protein Expression in Escherichia coli, Advances and Challenges. Frontiers in Microbiology, 5, 1-17.
[3] Hannig, G., and Makrides, S.C. (1998) Strategies for Optimizing Heterologous Protein Expression in Escherichia coli. Trends in Biotech, 16, 54-60.
[4] Baneyx, F. (1999) Recombinant Protein Expression in Escherichia coli. Current Opinion in Biotechnology, 10, 411- 421.
[5] Panda, A.K. (2003) Bioprocessing of Therapeutic Proteins from the Inclusion Bodies of Escherichia coli. Adv Biochem Eng Biotechnol, 85, 43-93.
[6] Palomares, L.A., Estrada-Mondaca, S. and Ramirez, O.T. (2004) Production of Recombinant Proteins, Challenges and Solutions. Methods in Molecular Biology, ch.2, 15-52.
[7] Mergulhao, F.J., Summers, D.K. and Monteiro, G.A. (2005) Recombinant Protein Secretion in Escherichia coli. Biotechnology Advances, 23, 177-202.
[8] Yin, J., Li, G., Ren, X. and Herrler, G. (2007) Select What You Need: A Comparative Evaluation of the Advantages and Limitations of Frequently Used Expression Systems for Foreign Genes. Journal of Bacteriology, 127, 335-347.
[9] Gilchrist, M.A. and Wagner, A. (2006) A Model of Protein Translation Including Codon Bias, Nonsense Errors, and Ribosome Recycling. Journal of Theoretical Biology, 239, 417-434.
[10] Kosiol, C., Holmes, I. and Goldman, N. (2007) An Empirical Codon Model for Protein Sequence Evolution. Molecular Biology and Evolution, 24, 1464-1479.
[11] Jukes, T.H. and Cantor, C.R. (1969) Evolution of Protein Molecules: Mammalian Protein Metabolism. 3. Academic Press, New York, 21-132.
[12] Kimura, M. (1980) A Simple Method for Estimating Evolutionary Rates of Base Substitutions through Comparative Studies of Nucleotide Sequences. Journal of Molecular Evolution, 16, 111-120.
[13] Bielawski, J.P. and Yang, Z.H. (2004) A Maxi-mum Likelihood Method for Detecting Functional Divergence at Individual Codon Sites, with Application to Gene Family Evolution. Journal of Molecular Evolution, 59, 121-132.
[14] Suzek, B.E., Ermolaeva, M.D., Schreiber, M. and Salzberg, S.L. (2001) A Probabilistic Method for Identifying Start Codons in Bacterial Genomes. Bioinformatics, 17, 1123-1130.
[15] Sato, T., Terabe, M., Watanabe, H., Gojobori, T., Ho-ri-Takemoto, C. and Miura, K. (2001) Codon and Base Biases after the Initiation Codon of the Open Reading Frames in the Escherichia coli Genome and Their Influence on the Translation Efficiency. Journal of Biochemistry, 129, 851-860.
[16] Shimomura, O., Johnson, F.H. and Saiga, Y. (1962) Extraction, Purification, and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea. Journal of Cellular and Comparative Physiology, 59, 223-239.
[17] Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. and Cormier, M.J. (1992) Primary Structure of the Aequorea victoria Green Fluorescent Protein. Gene, 111, 229-233.
[18] Miyawaki, A. and Tsien, R.Y. (2000) Monitoring Protein Conformations and Interactions by Fluorescence Resonance Energy Transfer between Mutants of Green Fluorescent Protein. Methods in Enzymology, 327, 472-500.
[19] Wu, Y.F., Zhou, Y.B., Song, J.P., Hu, X.J., Ding, Y. and Zhang, Z.H. (2008) Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystal-lization. Biochemistry and Molecular Biology Education, 36, 43-54.
[20] Poppenborg, L., Friehs, K. and Flaschel, E. (1997) The Green Fluorescent Protein is a Versatile Reporter for Bioprocess Monitoring. Journal of Biotechnology, 58, 79-88.
[21] Johnson, F.H., Shimomura, O., Saiga, Y., Gershman, L.C., Reynolds, G.T. and Waters, J.R., (1962) Quantum Efficiency of Cypridina Luminescence, with a Note on That of Aequorea. Journal of Cellular and Comparative Physiology, 60, 85-103.
[22] Morin, J.G. and Hastings, J.W. (1971) Energy Transfer in a Biolumi-nescent System. Journal of Cellular Physiology, 77, 313-318.
[23] Morise, H., Shimomura, O., Johnson, F.H. and Winant, J. (1974) Intermolecular Energy Transfer in the Bioluminescent System of Aequorea. Biochemistry, 13, 2656-2662.
[24] Tsien, R.Y. (1998) The Great Fluorescent Protein. Annual Review of Biochemistry, 67, 509-544.
[25] Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H. and Sheen, J. (1996) Engineered GFP as a Vital Reporter in Plants. Current Biology, 3, 325-330.
[26] Yang, F., Moss, G. and Maheswaran, G. (1996) The Molecular Structure of Green Fluorescent Protein. Nature Biotechnology, 14, 1246-1251.

Copyright © 2022 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.