FIELD: genetic engineering, molecular biology, medicine, endocrinology.
SUBSTANCE: invention relates, in particular, to preparing human proinsulin Lyspro and can be used for the development of medicinal preparations of new generation for treatment of insulin-dependent diabetes mellitus. Method involves construction of plasmid pLP-3-1 encoding hybrid polypeptide with the human proinsulin Lyspro sequence wherein sequence of domain B of staphylococcus protein A is connected through peptide linker HiS6Gly/SerArg with amino acid sequence of human proinsulin of molecular mass 3.3 MDa (5051 base pairs) comprising Hind III/Bam HI-fragment of plasmid pPINS07 including hybrid tac-promoter, β-lactamase gene (bla), region of replication start (ori), E. coli transcription operator of ribosome operon, nucleotide sequence encoding the sequence of amino acids in domain B of protein B in S. aureus containing synonymous change C → T at position 3 of the third codon GAC connected with nucleotide sequence encoding peptide His6GlySerArg, and Hind III/Bam HI-fragment (271 base pairs) encoding human proinsulin Lyspro, restriction sites with the following coordinates: Eco RI - 1, Nco I - 217, Bam HI - 221, Pst I - 342 and 417, Hind III - 492, Sal GI - 4513, Cla I - 4792. Microorganisms Escherichia coli are transformed with plasmid pPL-3-1 and the strain Escherichia coli TG-1/pLP-3-1 is obtained that is a producer of hybrid polypeptide with the human proinsulin Lyspro sequence. Invention allows preparing human proinsulin Lyspro with high yield by simplified technology.
EFFECT: valuable properties of plasmid and strain.
3 cl, 3 dwg, 4 ex
