RECOMBINANT PLASMID pET-GST-3CL-GPG PROVIDING SYNTHESIS OF SARS-CoV-2 3CL PROTEASE IN E. COLI CELLS IN SOLUBLE FORM Russian patent published in 2022 - IPC C12N15/50 C07K14/165 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A recombinant plasmid pET-GST-3CL-GPG is described, which provides the synthesis of SARS-CoV-2 3CL protease in E. coli cells in a soluble form, having the nucleotide sequence of SEQ ID NO: 2. The plasmid has a map shown in Fig. 1 and contains the following elements: AmpR promoter; a genetic marker that determines ampicillin resistance of E. coli bacteria cells transformed with the recombinant AmpR plasmid; origin of replication site ori; lactose repressor lacI; DNA-binding protein that inhibits the expression of E. coli genes and ensures the expression of the target recombinant 3CL-GPG-6His protein after the addition of an isopropyl-β-D-1-thiogalactopyranoside (IPTG) inducer; lacI promoter; the T7 phage promoter, which ensures the expression of the GST-3CL-GPG gene sequence in E. coli cells; lac operator; RBS; gene GST-glutathione-S-transferase from Scihistosoma japonicum; Pep - region encoding the GSTSAVLQ peptide; the SARS-CoV-2 chimeric 3CL gene also including the GPG and 6His sequence, having the nucleotide sequence of SEQ ID NO: 1, and encoding the synthesis of the 3CL-GPG-6His protease with the amino acid sequence of SEQ ID NO: 3; T7 terminator.

EFFECT: invention can be used in biotechnology to obtain the 3CL enzyme, which is the main protease of SARS-CoV-2. This ensures the synthesis and transport of the chimeric variant of the 3CL protein into the periplasmic space of E. coli in a soluble form.

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