Biomedical Chemistry: Research and Methods 2021, 4(3), e00091

Expression and Isolation of N-Terminal Truncated Human Recombinant Renalase in Prokaryotic Cells

V.I. Fedchenko *, A.A. Kaloshin, S.A. Kaloshina, A.E. Medvedev

Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia; *e-mail: valfed38@yandex.ru

Keywords: renalase; protein expression; inclusion bodies; protein solubilization; isolation

DOI:10.18097/BMCRM00158

The whole version of this paper is available in Russian.

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 1.6.3.5). 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.
Figure 1. Analysis of the protein products obtained after expression of the vector pET-hRen(-sp) in Е. coli Rosetta (DE3) cells. Tracks: 1 – molecular weight protein markers (arrows indicate protein markers of 70 kDa, 55 kDa, 45 kDa, 35 kDa, and 25 kDa. 2 – proteins produced by the cells without IPTG stimulation; 3 – proteins produced by IPTG stimulated cells; 4 – inclusion bodies containing recombinant tRNLS; 4 – chromatographically purified tRNLS.
Figure 2. Analysis of recombinant tRNLS solubilization from inclusion bodies in the buffer containing 8 M urea. Here and in the subsequent figure a – supernatant after inclusion bodies solubilization and centrifugation; b – sediment after inclusion bodies solubilization and centrifugation. Tracks: 1 – incubation for 15 min; 2 – incubation for 30 min; 3 – incubation for 1 h; 4 – incubation for 12 h.
Figure 3. Analysis of recombinant tRNLS solubilization from inclusion bodies in the buffer containing 6 М GdnHCl.

FUNDING

This work was supported by a grant from the Russian Foundation for Basic Research (project number 20–015–00104).

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