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.

Figure 1

The schematic diagram illustrating the construction of a full-length coding sequence of hRenalases. Step1: Seven individual exons of hRenalase1 (1ExRe1/7ExRe7) with overlapping sequences (OS) were amplified by means of corresponding forward (Pr-f) and reverse (Pr-r) primers; Step2: The amplified exons were sequentially linked together by overlapping PCR. (a) 1ExRe1 with 2ExRe1, 3ExRe1 with 4ExRe1, 5ExRe1 with 6ExRe1; (b) 1/2ExRe1 with 3/4ExRe1 and exons 5/6ExRe1 with 7ExRe1; (c) 1/4ExRe1 with 5/7ExRe1.

Figure 2

Amplification of individual exons of hRenalase1. (A) Agarose gel electrophoresis of PCR products of hRenalase1 exons obtained using the human genomic DNA; (B) Step 1 of PCR amplification of hRenalase1 exons using human genomic DNA as a template. The calculated sizes of the amplified exons (corrected for the presence of the OS sequences) were: 1—1ExRe1 (131 bp); 2—2ExRe1 (126 bp); 3—3ExRe1 (166 bp); 4—4ExRe1 (179 bp); 5—5ExRe1 (193 bp); 6—6ExRe1 (197 bp); 7—7ExRe1 (188 bp). L—DNA ladder of 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp. Here and in subsequent figures all designations are the same as in Figure 1.

Figure 3

Joining of hRenalase1 exons into the full-length coding sequence. Agarose gel electrophoresis of PCR products of hRenalase1 exons obtained during Step 2. The calculated sizes of the amplified exons (corrected for the presence of the OS sequences) were: 1—1–2ExRe1 (236 bp); 2—3–4ExRe1 (325 bp); 3—5–6ExRe1 (370 bp); 4—1/4ExRe1 (538 bp); 5—5/7ExRe1 (535 bp); 6—1/7ExRe1 (1061 bp). L—DNA ladder (50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp); L-2—DNA ladder-2 (50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp).

Figure 4

Nucleotide sequences of hRenalase1 ORF available from GenBank (locus NM_001031709.1; upper sequence). hRenalase1 ORF (median sequence) obtained in this study by sequential exon joining, and amino acid sequence of hRenalase1 (lower sequence, a one-letter code). SNPs are shown in bold capital letters.

Figure 5

Expression of hRenalase1 and hRenalase2 in E. coli cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of expression of polyHis hRenalase1 (RenI—39 kDa) and hRenalase2 (RenII—36 kDa) in E. coli Rosetta (DE3) cells transformed by pET-hRenI and pET-hRenII vectors. Tracks 1 and 4—polyHis hRenalase2 (RenII—36 kDa) and hRenalase1 (RenI—39 kDa), respectively, purified on Ni-Sepharose; Tracks 2 and 5—lysates of cells transformed pET-hRenI и pET-hRenII induced with 1.0 mM IPTG; Tracks 3 and 6—lysates of cells transformed pET-hRenI and pET-hRenII, without IPTG; Track M—molecular mass markers. Mass values are shown on the right.

Figure 6

Western blot analysis of hRenalase1 and hRenalase2 using sheep polyclonal antibodies against hRenalase1. (A) Staining of the nitrocellulose membrane with Ponceau S after electrotransfer from the SDS-PAGE gel; Track 1—polyHis hRenalase1 (RenI—39 kDa); Track 2—polyHis hRenalase2 (RenII—36 kDa); Track M—molecular mass marker; mass values are shown on the left; (B) Western blot analysis with sheep polyclonal antibodies against hRenalase1.