Construction of a strain-producer of the chimeric protein consisting of RNA polymerase and a DNA-affinity domain
https://doi.org/10.29235/1561-8323-2018-62-5-601-607
Abstract
One of the recent perspective trends of molecular biotechnology is cell-free synthesis of protein. The procedure of cell-free synthesis of protein is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes. Procreation of the transcription stage requires participation of specific RNA polymerase which initiates process of mRNA synthesis from the particular sites of recognition. Often the DNA-dependent RNA polymerase of a bacteriophage of T7 (T7 RNA polymerase) is for this purpose applied. For improvement of qualitative characteristics of the T7 RNA polymerase in the real work the new strain of Escherichia coli producing this enzyme fused with the DNA-affine Sso7d domain of a thermophilic bacterium Sulfolobus solfataricus is created. The producing ability of the received recombinant strain concerning synthesized chimera protein reaches 625 un/l of cultural liquid, and the specific activity of the purified enzyme preparation was 80 un/ μg of protein. The received enzyme is intended for use as tools at synthesis of proteins in cell-free system.
About the Authors
I. S. Kazlovskiy
National Academy of Sciences of Belarus, Institute of Microbiology
Belarus
Belarus
Kazlovskiy Il ’ya Sergeevich - Master of Biology, Junior researcher.
2, Kuprevich Str., 220141, Minsk
M. A. Zinchenko
National Academy of Sciences of Belarus, Institute of Microbiology
Belarus
Belarus
Zinchenko Anatoliy Ivanovich - Corresponding Member, D. Sc. (Biology), Professor, Head of the Laboratory.
2, Kuprevich Str., 220141, Minsk
References
1. Bundy B. C., Franciszkowicz M. J., Swartz J. R. Escherichia coli-based cell-free synthesis of virus-like particles. Biotechnology and Bioengineering, 2008, vol. 100, no. 1, pp. 28-37. https://doi.org/10.1002/bit.21716
2. Bundy B. C., Swartz J. R. Efficient disulfide bond formation in virus-like particles. Journal of Biotechnology, 2011, vol. 154, no. 4, pp. 230-239. https://doi.org/10.1016/jjbiotec.2011.04.011
3. Smith M. T., Varner C. T., Bush D. B., Bundy B. C. The incorporation of the A2 protein to produce novel QP virus-like particles using cell-free protein synthesis. Biotechnology Progress, 2012. vol. 28, no. 2, pp. 549-555. https://doi.org/10.1002/btpr.744
4. Hovijitra N. T., Wuu J. J., Peaker B., Swartz J. R. Cell-free synthesis of functional aquaporin Z in synthetic liposomes. Biotechnology and Bioengineering, 2009, vol. 104, no. 1, pp. 40-49. https://doi.org/10.1002/bit.22385
5. Georgi V., Georgi L., Blechert M., Bergmeister M., Zwanzig M., Wustenhagen D. A., Bier F. F., Jung E., Kubick S. On-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode. Lab on a Chip, 2016, vol. 16, no. 2, pp. 269-281. https://doi.org/10.1039/c5lc00700c
6. Caschera F., Noireaux V. Preparation of amino acid mixtures for cell-free expression systems. Biotechniques, 2015, vol. 58, no. 1, pp. 40-43. https://doi.org/10.2144/000114249
7. Roberts J. W. Termination factor for RNA synthesis. Nature, 1969, vol. 224, no. 5225, pp. 1168-1174. https://doi.org/10.1038/2241168a0
8. Studier F. W. T7 expression systems for inducible production of proteins from cloned genes in E. coli. Current Protocols in Molecular Biology, 2018, vol. 124, no. 1, e63. https://doi.org/10.1002/cpmb.63
9. Kazlovskii I. S., Pymko A. N., Kvach S. V., Zinchenko A. I. Construction of Escherichia coli strain, producing bacteriophage T7 RNA polymerase. Mikrobnye biotechnologii: fundamental’nye i prikladnye aspekty [Microbial biotechnology: fundamental and applied aspects]. Minsk, Belaruskaya navuka Publ., 2016, vol. 8, pp. 72-81 (in Russian).
10. Wang Y., Prosen D. E., Mei L., Sullivan J. C., Finney M., Vander Horn P. B. A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research, 2004, vol. 32, no. 3, pp. 1197-1207. https://doi.org/10.1093/nar/gkh271
11. Korovashkina A. S., Kvach S. V., Eroshevskaya L. A., Zinchenko A. I. Production of thermostable DNA polymerase suitable for whole-blood polymerase chain reaction. Varfolomeev S. D., Zaikov G. E., Krylova L. P. (eds.). Biochemistry and Biotechnology: Research and Development. New York, Nova Science Publishers, Inc., 2012, pp. 1-5.
12. Quan J., Tian J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS ONE, 2009, vol. 4, no. 7, e6441. https://doi.org/10.1371/journal.pone.0006441
13. You C., Zhang X. Z., Zhang Y. H. P. Simple cloning via direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis. Applied and Environmental Microbiology, 2012, vol. 78, no. 5, pp. 1593-1595. https:// doi.org/10.1128/aem.07105-11
14. Arnaud N., Cheynet V, Oriol G., Mandrand B., Mallet F. Construction and expression of a modular gene encoding bacteriophage T7 RNA polymerase. Gene, 1997, vol. 199, no. 1-2, pp. 149-156. https://doi.org/10.1016/s0378-1119(97)00362-4
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