SOLUBLE RECOMBINANT PLASMID PET-GST-3CL ENSURING SYNTHESIS OF 3CL SARS-COV-2 PROTEASE IN E. COLI CELLS Russian patent published in 2023 - IPC C12N15/50 

FIELD: biotechnology.

SUBSTANCE: described is a soluble recombinant plasmid pET-GST-3CL ensuring the synthesis and transport of chimeric protease 3CL SARS-CoV-2 in the periplasmic space of E. coli cells, with the nucleotide sequence SEQ ID NO: 2, a size of 6981 bps, containing, in accordance with the physical and genetic map represented in Fig. 1, the following elements: - an AmpR promoter with the coordinates from 494 to 598 bps; - a genetic marker determining the resistance to ampicillin of the bacterial cells of E. coli transformed with recombinant plasmid AmpR, with coordinates from 599 to 1459 bps; - an origin of replication ori with the coordinates from 1630 to 2218 bps; - lacI - a DNA-binding protein inhibiting the expression of E. coli genes, ensuring the expression of the target recombinant protein 3CL after adding the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG), with the coordinates from 3648 to 4730 bps; - lacI promoter with the coordinates from 4731 to 4808 bps; a T7 phage promoter with the coordinates from 5117 to 5135 bps, ensuring the expression of the gene sequence GST-3CL in E. coli cells; a lac operator with the coordinates from 5136 to 5160 bps; - RBS - the ribosome binding site with the coordinates from 5191 to 5196 bps; - the GST gene - glutathione-S-transferase from Schistosoma japonicum with the coordinates from 5205 to 5857 bps; - Pep - a site encoding the peptide GSTSAVLQ, with the coordinates from 5859 to 5882 bps; - a chimeric gene 3CL SARS-CoV-2 with the nucleotide sequence SEQ ID NO: 1 and the coordinates from 5883 to 6806 bps, encoding the synthesis of a 3CL protease protein with the amino acid sequence SEQ ID NO: 3; - a T7 terminator with the coordinates from 6909 to 6956 bps.

EFFECT: invention expands the range of means for producing the 3CL protein.

1 cl, 3 dwg, 1 tbl, 5 ex