Biomedical Chemistry: Research and Methods 2022, 5(2), e00175

Construction and Expression of the Chimeric Human Renalase Gene Encoding the N-Terminal Signal Sequence of the Secretory Protein Prolactin

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

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

Keywords:PCR; cloning; expression; renalase; prolactin; signal sequence

DOI:10.18097/BMCRM00175

The whole version of this paper is available in Russian.

Renalase (RNLS) is a protein that performs various protective functions both inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase (EC 1.6.3.5). Extracellular RNLS lacking an N-terminal peptide does not interact with FAD and exhibits various protective effects on the cell through interaction with receptor proteins. The mechanisms and factors responsible for RNLS transport out of the cell are not fully understood. It is well known that the signal sequence plays a key role in the classical mechanism of protein transport outside cells. One of the approaches to study the secretion of RNLS from the cell can be the creation of chimeric forms of the protein with a modified N-terminal amino acid signal sequence. Bioinformatics analysis showed that the signal sequence of the prolactin gene (PRL), connected to the template sequence of the RNLS gene, gave the classic signal characteristic of secretory proteins. On this basis, this paper describes: (i) a method for constructing the human RNLS gene in which the N-terminal sequence encoded by the RNLS gene was replaced by the N-terminal sequence encoded by the PRL gene; (ii) expression of this chimeric genetic construct.
Figure 1. Messenger RNA (mRNA) isomers of the PRL gene. mRNA variant 1 contains 894 nucleotides and includes a coding region (CDS) of 55-738 nucleotides, which contains 84 nucleotides encoding a signal peptide (SP) and 600 nucleotides encoding matrix protein (MP). mRNA variant 2 contains 1013 nucleotides and a CDS of 174-857 nucleotides, which contains 84 nucleotides, encoding SP and 600 nucleotides encoding MP. mRNA variant X1 contains 1016 nucleotides and CDS of 174-860 nucleotides, which contains 87 nucleotides encoding SP and 600 nucleotides encoding MP. mRNA variant X2 contains 1029 nucleotides and CDS of 187-873 nucleotides, which contains 84 nucleotides encoding SP and 600 nucleotides encoding MP.
Figure 2. Agarose gel electrophoresis (2%) of DNA fragments from PCR reactions (left to right). M1) - DNA markers: 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp .o., 900 bp, 1000 bp. 1). - 1092 bp, chimeric DNA (a DNA fragment of the PRL gene connected to a fragment of the matrix DNA RNLS. 2). - 990 bp, a fragment of the matrix DNA of the RNLS gene. 3). - 120 bp, DNA fragment of the PRL gene. M2) - DNA markers: 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp .o., 450 bp, 500 bp.
Figure 3.

Electrophoresis in 12% PAAG of E.coli cell proteins during expression of the chimeric RNLS gene (vector pET-PRL-RenI): 1 - total protein of E. coli cells (DE3 Rosetta strain) without induction; 2 – total protein after induction of IPTG cells; 3 – RNLS protein with the signal sequence of the PRL gene (RNLS-prl), after purification on a Ni-Sepharose column.

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Table 1. Prediction of the presence of the signal peptide (SP) in chimeric renalases.

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Table 2. Prediction of cellular localization of renalase with the N-terminal peptide from other proteins.

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Table 3. Primers used in PCR of the chimeric RNLS gene .

FUNDING

This work was supported by the Russian Foundation for Basic Research (project no. 20–015–00104).

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