Expression and Isolation of N-Terminal Truncated Human Recombinant Renalase in Prokaryotic Cells
Keywords:isolation; protein solubilization; inclusion bodies; protein expression; renalase
Renalase (RNLS) is a flavoproteinin which its N-terminal peptide (residues 1-17) has several important functions. In cells, it participates in the formation of the so-called Rossmanfold (residues 2-35), needed for «accommodation» of the FAD cofactor and for performing the catalytic functions of RNLS as a FAD-dependent oxidoreductase (EC 220.127.116.11). RNLS secretion into the extracellular space is accompanied by cleavage of this peptide. The resultant truncated extracellular RNLS cannot bind FAD and therefore performs various noncatalytic functions. In this work, we have performed expression the genetic construct encoding RNLS lacking its N-terminal signal peptide (tRNLS) in E. coli Rosetta (DE3) cells. The recombinant protein was accumulated in inclusion bodies in an insoluble form, which could be solubilized in the presence of a high concentration of urea or guanidine chloride. In contrast to full-length RNLS, which was effectively solubilized in the presence of 8 M urea, tRNLS was preferentially solubilized in the presence of 6 M guanidine chloride.
- Xu J., Li G., Wang P., Velazquez H., Yao X., Li Y, Wu Y., Peixoto A., Crowley S., Desir G.V. (2005) Renalase is a novel, soluble monoamine oxidase that regulates cardiac function and blood pressure. J. Clin. Invest.,115, 1275–1280. DOI
- Milani M., Ciriello F., Baroni S., Pandini V., Canevari G.,Bolognesi M., Aliverti A. (2011) FAD-binding site and NADP reactivity in human renalase: a new enzyme involved in blood pressure regulation. J. Mol. Biol., 411(2), 463-473. DOI
- Fedchenko V.I., Buneeva O.A., Kopylov A.T., Veselovsky A.V., Zgoda V.G., Medvedev A.E. (2015) Human urinary renalase lacks the N-terminal signal peptide crucial for accommodation of its FAD cofactor. International Journal of Biological Macromolecules, 78, 347–353. DOI
- Moran G.R., Hoag M.R. (2017) The enzyme: Renalase. Arch. Biochem. Biophys.,632, 66-76. DOI
- Fedchenko V., Kopylov A., Kozlova N., Buneeva O., Kaloshin A., ZgodaV., Medvedev A. (2016) Renalase secreted by human kidney HEK293T cells lacks its N-terminal peptide: implications for putative mechanisms of renalase action. Kidney Blood Press. Res., 41, 593-603. DOI
- Medvedev A.E., Veselovsky A.V., Fedchenko V.I. (2010) Renalase, a new secretory enzyme responsible for selective degradation of catecholamines: achievements and unsolved problems. Biochemistry (Moscow), 75(8), 951-958. DOI
- Severina I.S., Fedchenko V.I., Veselovsky A.V., Medvedev A.E.(2015) The history of renalase from amine oxidase to a α-NAD(P)H-oxidase/anomerase. Biomed Khim. 2015,61(6), 667-679. DOI
- Wang Y., Safirstein R., Velazquez H., Guo X.J., Hollander L., Chang J., Chen T.M., Mu J.J., Desir G.V. (2017) Extracellular renalase protects cells and organs by outside-in signalling. J. Cell Mol. Med., 21(7), 1260-1265. DOI
- Pointer T.C., Gorelick F.S., Desir G.V.(2021) Renalase: a multi-functional signaling molecule with roles in gastrointestinal disease. Cells, 10(8), 2006. DOI
- Wang, R.P. peptide Wang L., Velazquez H., Chang J., Safirstein R., Desir G.V. (2015) Identification of a receptor for extracellular renalase. PLoS One, 10, e0122932. DOI
- Fedchenko V.I., Kaloshin A.A., Kozlova N.I., Kopylov, A.T., Medvedev A.E.(2020) Construction of a chimeric human gene encoding renalase with a modified N-terminus. Biomedical Chemistry: Research and Methods, 3(3), e00137. DOI
- Fedchenko V. I., Kaloshin A.A., Mezhevikina L.M., Buneeva O.A., Medvedev A.E. (2013) Construction of the coding sequence of the transcription variant 2 of the human renalase gene and its expression in the prokaryotic system. Int. J. Mol. Sci. 14, 12764-12779. DOI
- Laemmli, U.K. (1970) Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature; 227, 680-685. DOI
- Kushner, S. R. (1978). An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In: Genetic engineering (Boyer H.B. and Nicosia S., eds.), p. 17, Elsevier/North-Holland, Amsterdam.
- Cohen, S.N., Chang, A.C.Y., Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Esherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA, 69, 2110-2114.
- Fedchenko V.I., Kaloshin A.A. (2019) A simplified method for obtainingcDNA of low-copy and silent eukaryotic genes using human renalase as anexample. Biomedical Chemistry: Research and Methods. 2(2), e00101. DOI
- Khomenko V.A., Sidorin E.V., Bakholdina S.I., Naberezhnykh G.A., Kim N.Y., Stenkova A.M., Chernysheva N.Y., Isaeva M.P., Solov'eva T.F. (2019) Inclusion Bodies of Recombinant OmpF Porin from Yersinia pseudotuberculosis: Properties and Structural Characterization. Biochemistry (Mosc), 84(6), 672-685. DOI
- Park A.R., Jang S.W., Kim J.S., Park Y.G., Koo B.S., Lee H.C. (2018) Efficient recovery of recombinant CRM197 expressed as inclusion bodies in E. coli. PLoS One. 3(7), e0201060. DOI
- Pandini V., Ciriello F., Tedeschi G., Rossoni G.; Zanetti G., Aliverti A. (2010) Synthesis of human renalase1 in Escherichia coli and its purification as a FAD-containing holoprotein. Protein Expr. Purif., 72, 244–253.
- Eiberle, M.K.; Jungbauer, A. (2010) Technical refolding of proteins: Do we have freedom to operate? Biotechnol. J. 5(6), 547-559. DOI
- Gautam S., Dubey P., Rather G.M., Gupta M.N. (2012) Non-chromatographic strategies for protein refolding. Recent Pat Biotechnol., 6(1), 57-68. DOI
- Machold C., Schlegl R., Buchinger W., Jungbauer A. (2005) Matrix assisted refolding of proteins by ion exchange chromatography. J. Biotechnol. 117, 83-97. DOI
- Clark E.D.B.(2001) Protein refolding for industrial processes. Curr. Opin. Biotechnol., 12, 207-202
- Marston F.A.O. (1986) The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem. J., 240, 1-12.