Construction of the Coding Sequence of the Transcription Variant 2 of the Human Renalase Gene and Its Expression in the Prokaryotic System

Abstract

Renalase is a recently discovered protein, involved in regulation of blood pressure in humans and animals. Although several splice variants of human renalase mRNA transcripts have been recognized, only one protein product, hRenalase1, has been found so far. In this study, we have used polymerase chain reaction (PCR)-based amplification of individual exons of the renalase gene and their joining for construction of full-length hRenalase2 coding sequence followed by expression of hRenalase2 as a polyHis recombinant protein in Escherichia coli cells. To date this is the first report on synthesis and purification of hRenalase2. Applicability of this approach was verified by constructing hRenalase1 coding sequence, its sequencing and expression in E. coli cells. hRenalase1 was used for generation of polyclonal antiserum in sheep. Western blot analysis has shown that polyclonal anti-renalase1 antibodies effectively interact with the hRenalase2 protein. The latter suggests that some functions and expression patterns of hRenalase1 documented by antibody-based data may be attributed to the presence of hRenalase2. The realized approach may be also used for construction of coding sequences of various (especially weakly expressible) genes, their transcript variants, etc.

Renalase Secreted by Human Kidney HEK293T Cells Lacks its N-Terminal Peptide: Implications for Putative Mechanisms of Renalase Action

“…The latter was obtained by amplification of the corresponding nucleotide sequence of the plasmid pET-hRenI, in which the renalase-1 gene contained an additional sequence coding a hexahistidine tag [14]. The renalase gene coding sequence was inserted by BamHI/XhoI restriction sites.…”

“…A custom polyclonal sheep antibody was raised against human recombinant renalase1 and purified by Pocard Ltd. (Moscow, Russia). A monoclonal anti-rabbit/sheep IgG antibody conjugated with horseradish peroxidase was from IMTEK (Moscow, Russia) (see also [14]). …”

“…Immunoblotting was performed by the method of Gallagner et al [31] as described in Fedchenko et al [14] using sheep antibody to renalase 1 and horseradish peroxidase conjugate with rabbit anti-sheep immunoglobulin.…”

“…Under these conditions transfected HEK293T cells were incubated for 30 h. After incubation, the cultivation medium was harvested, cleared by low speed centrifugation (1000 rpm for 5 min; Heraeus Megafuge 40 centrifuge; Thermo Scientific USA) and filtered through a sterile PVDFFilter with a pore diameter of 0.45 m (Millipore). Based on renalase isolation from human urine samples [14] the protein concentrated by 75% ammonium sulfate precipitation was then dissolved in 50 mM TrisHCl buffer, containing 5 mM EDTA, pH 8.0, and stored at -20°C. The cell monolayer was mechanically scraped off from the substrate, washed with saline, and sedimented at 5000 rpm for 10 min using the Eppendorf centrifuge 5415R.…”

“…All known renalases synthesized in eukaryotic and prokaryotic cells contain cleavable N-terminal signal peptide (residues 1-17) [3][4][5][6][7][12][13][14] required for extracellular secretion of renalase (Fig. 1).…”