Abstract
Protein splicing is a post-translational autocatalytic process that results in the excision of an internal peptide (the intein) from a precursor protein and the ligation of the flanking protein sequences (the exteins). The high specificity of intein-mediated excision of protein precursors permits the use of protein splicing in biotechnology. This work was aimed the production of human growth hormone with a native N-terminus in E. coli. A chimeric protein consisting of a short N-terminal peptide, the Mxe GyrA intein, and human growth hormone was constructed. The formyl-methionine modified N-terminal peptide was intended for removal via splicing during translation. This intein has been shown to mediate the cleavage of exteins, but their subsequent ligation has never been observed. This permitted the production of human growth hormone with the native N-terminus. The effect of various factors on cleavage efficiency was also studied. The most efficient cleavage of the chimeric protein (60–80%) was observed in the presence of an inductor (100 mM β-mercaptoethanol) upon incubation for 4–6 days.
Similar content being viewed by others
References
Datta, B., MAPs and POEP of the Roads from Prokaryotic to Eukaryotic Kingdoms, Biochimie, 2000, vol. 82, pp. 95–107.
Wood, D., Simplified Protein Purification Using Engineered Self-Cleaving Affinity Tags, J. Chem. Tech. Biotech., 2003, no. 78, pp. 103–110.
Chong, S., Montello, G., Zhang, A., Cantor, E., Liao, W., Xu, M., and Benner, J., Utilizing the C-terminal Cleavage Activity of a Protein Splicing Element to Purify Recombinant Proteins in a Single Chromatographic Step, Nucl. Acids Res., 1988, vol. 26, no. 22, pp. 5109–5115.
Kane, P.M., Yamashiro, C.T., Wolczyk, D.F., Neff, K., Goebl, M., and Stevens, T.H., Protein Splicing Converts the Yeast TFPl Gene Product to the 69 kDa Subunit of the Vacuolar H+-adenosine Triphosphatase, Science, 1990, vol. 250, pp. 651–657.
Yu, R., Hong, A., Dai, Y., and Gao, Y., Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide, Acta Biochim. Biophys. Sin., 2004, vol. 36, no. 11, pp. 759–766.
Starokadomskii, P.L., Protein Splicing, Ukrainian Biochem. J., 2005, no. 4, pp. 14–29.
Noren, C., Wang, J., and Perler, F., Dissecting the Chemistry of Protein Splicing and Its Applications, Angew. Chem. Int. Ed., 2000, vol. 39, pp. 450–466.
Maniatis, T., Fritch, E., and Senbrooke, S, J., Methods of Genetic Engineering. Molecular Cloning, Moscow: Mir, 1984, p. 480.
Novagen pET System Manual. 11th Ed. EMD Biosciences, Darmstadt (Germany), 2005, p. 80.
Affinity chromatography: Methods. Din, P., Johnson, U., and Middle, F., Eds., Moscow: Mir, 1988, pp. 98–99.
Westermeier, R., Electrophoresis in Practice, Weinheim: VCH, 1997, p. 331.
Telenti, A., Southworth, M., Alcaide, F., Daugelat, S., Jacobs, W.R., and Perler, F.B., The Mycobacterium xenopi GyrA Protein Splicing Element: Characterization of a Minimal Intein, J. Bacteriol., 1997, vol. 179, no. 20, pp. 6378–6382.
Perler, F. In Base, the Intein Database, Nucleic Acids Res., 2002, vol. 30, pp. 383–384.
Klabunde, T., Sharma, S., Telenti, A., Jacobs, W., and Sacchettini, J., Crystal Structure of GyrA Intein from Mycobacterium xenopi Reveals Structural Basis of Protein Splicing, Nature Struct. Biol., 1998, vol. 5, no. 1, pp. 31–36.
Rabinovich, V.A. and Khavin, Z.Ya., Short Chemical Handbook, 2nd edition, Leningrad: Khimiya, 1978, p. 240.
Brocklehurst, K., Stuchbury, T., and Malthouse, J., Reactivity of Neutral and Cationic Forms of 2, 2′-dipyridyl Disulfide Towards Thiolate Anions, Biochem. J., 1979, vol. 183, pp. 233–238.
Author information
Authors and Affiliations
Additional information
Original Russian Text © P.L. Starokadomskyy, I.Ya. Dubey, O.V. Okunev, D.M. Irodov, 2007, published in Tsitologiya i Genetika, 2007, Vol. 41, No. 2, pp. 3–11.
About this article
Cite this article
Starokadomskyy, P.L., Dubey, I.Y., Okunev, O.V. et al. Construction of a chimeric intein-containing protein and the search for conditions for its cleavage. Cytol. Genet. 41, 69–75 (2007). https://doi.org/10.3103/S0095452707020016
Received:
Issue Date:
DOI: https://doi.org/10.3103/S0095452707020016