Biomedical Chemistry: Research and Methods 2024, 7(2), e00228

Generation of C-Terminal Sequences of Human Renalase-1 and Renalase-2 Encoded by Alternative Exons

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

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

Keywords:renalase (RNLS); alternative exons of the RNLS gene; RNLS1 and RNLS2 isoforms of renalase; cloning; expression; protein purification

DOI:10.18097/BMCRM00228

The whole version of this paper is available in Russian.

A method for generation of C-terminal amino acid sequences fused to dihydrofolate reductase (DHFR) and specific for RNLS1 and RNLS2 isoforms of renalase is described. It includes synthesis of nucleotide sequences of alternative exons of RNLS1-9ex and RNLS2-10ex, determining the differences in the primary structure of these proteins, their fusion with the coding sequence of DHFR and expression of these genetic constructs in cells of the E. coli Rosetta cells. Chromatographic purification on a column containing Ni Sepharose resulted in highly purified preparations of reombinant ReI-9ex and ReII-10ex proteins with an electrophoretic purity of about 95%.
Figure 1. Exon organization of the RNLS gene (NC_000010.11). The location of 10 exons of the RNLS gene is shown with their lengths (782 bp, 106 bp, 143 bp, 159 bp, 174 bp, 285 bp, 176 bp, 90 bp, 1409 bp and 1169 bp). The RNLS1 mRNA transcriptional variant of 2420 nucleotides in length (NM_001031709.2) includes exons 1, 2, 3, 4, 5, 7, and 9. The RNLS2 mRNA transcriptional variant of 2180 nucleotides in length (NM_018363.3) includes exons 1, 2, 3, 3, 4, 5, 7, and 10.
Figure 2. Electrophoresis in 2% agarose gel of DNA amplicons. 1- DNA amplicon of 147 bp from exon 9. 2 - DNA amplicon 87 bp from exon10. M - DNA marker: 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp.
Figure 3. The scheme of RNLS1-9ex and RNLS2-10ex cloning into the pQE40 vector by BglII and PstI restriction sites. Explanations are given in the text.
Figure 4. Control expression of the pQE-ReI-9ex and pQE-ReII-10ex vectors in E. coli Rosetta cells in eight clones. A-(clones 1-8) - total protein of E. coli cells after IPTG induction in clones containing the pQE-ReI-9ex vector. B-(clones 1-8) - total protein of E. coli cells after IPTG induction in clones containing the pQE-ReII-9ex vector. M is the molecular mass marker of proteins. Protein molecular masses of 75 kDa, 60 kDa, 37 kDa, and 25 kDa are indicated on the left. On the right, the size of the recombinant ReI-9ex protein with an estimated mass of 28.5 kDa and the recombinant ReII-10ex protein with an estimated molecular mass of 25.6 kDa are indicated, respectively.
Figure 5. Electrophoresis of recombinant cell proteins in 12% PAAG. M is the molecular mass marker. Protein molecular masses of 75 kDa, 60 kDa, 37 kDa, and 25 kDa are indicated on the left. 1 - ReI-9ex protein with a calculated molecular mass of 28.5 kDa after purification on a Ni-Sepharose column; 2 - ReII-10ex protein with a calculated molecular mass of 25.6 kDa.

CLOSE
Table 1. Primers used for PCR-based amplification of RNLS-1-9ex и RNLS-2-10ex

FUNDING

The work was performed within the framework of the Program for Basic Research in the Russian Federation for a long-term period (2021-2030). (no. 122030100170-5).

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