Method for producing human recombinant insulin

A method of recombinant human insulin production by means of construction of recombinant plasmid DNA, which encode proinsulin, the development and culturing of Escherichia coli strain producing hybrid polypeptide, cell separation and disruption and polypeptide isolation, its enzymatic conversion followed by purification and isolation of desired product, a method which differ in that way, that recombinant plasmid DNA fragment which encode hybrid polypeptide comprising amino acid sequence of human proinsulin is a part of expression vector pIK8-proins, or pMUT12, or pISYN2, or pCIM61, or pDIM07 or hybridize to one of the foregoing DNA inserts and which code on expression for human proinsulin precursor, so that recombinant plasmid DNA fragment which encode hybrid polypeptide comprising human proinsulin sequence and which is a part of pIK8-proins plasmid has the following sequence:

• 1
• ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCAT
• 1
• M G S S H H H H H H S S G L V P R G S H
• 61
• ATGCGCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTG
• 21
• M R F V N Q H L C G S H L V E A L Y L V
• 121
• TGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAG
• 41
• C G E R G F F Y T P K T R R E A E D L Q
• 181
• GTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTG
• 61
• V G Q V E L G G G P G A G S L Q P L A L
• 241
• GAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTC
• 81
• E G S L Q K R G I V E Q C C T S I C S L
• 301
• TACCAGCTGGAGAACTACTGCAACTAG
• 101
• Y Q L E N Y C N *,
• recombinant plasmid DNA fragment which encode hybrid polypeptide comprising human proinsulin sequence and which is a part of pMUT12 plasmid has the following sequence:
• 1
• ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCAT
• 1
• M G S S H H H H H H S S G L V P R G S H
• 61
• ATGCGCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTG
• 21
• M R F V N Q H L C G S H L V E A L Y L V
• 121
• TGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCAAGCGTGGCATTGTGGAACAATGC
• 41
• C G E R G F F Y T P K T K R G I V E Q C
• 181
• TGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAG
• 61
• C T S I C S L Y Q L E N Y C N *,
• recombinant plasmid DNA fragment which encode hybrid polypeptide comprising human proinsulin sequence and which is a part of pISYN2 plasmid has the following sequence:
• 1
• ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCAT
• 1
• M G S S H H H H H H S S G L V P R G S H
• 61
• ATGCGCTTTGTGAACCAGCATCTGTGTGGCAGCCACCTGGTGGAAGCGCTGTATTTAGTG
• 21
• M R F V N Q H L C G S H L V E A L Y L V
• 121
• TGCGGCGAGCGTGGCTTCTTTTATACCCCGAAAACCAAACGTGGCATTGTGGAACAGTGT
• 41
• C G E R G F F Y T P K T K R G I V E Q C
• 181
• TGCACCAGTATTTGTAGCCTGTATCAGCTGGAAAATTACTGCAACTAA
• 61
• C T S I C S L Y Q L E N Y C N *,
• recombinant plasmid DNA fragment which encode hybrid polypeptide comprising human proinsulin sequence and which is a part of pCIM61 plasmid has the following sequence:
• 1
• ATGCGAAAGAAGCGGAAGAAGAAGCGTTTTGTGAACCAGCATCTGTGTGGCAGCCACCTG
• 1
• M R K K R K K K R F V N Q H L C G S H L
• 61
• GTGGAAGCGCTGTATTTAGTGTGCGGCGAGCGTGGCTTCTTTTATACCCCGAAAACCAAA
• 21
• V E A L Y L V C G E R G F F Y T P K T K
• 121
• CGTGGCATTGTGGAACAGTGTTGCACCAGTATTTGTAGCCTGTATCAGCTGGAAAATTAC
• 41
• R G I V E Q C C T S I C S L Y Q L E N Y
• 181
• TGCAACTAA
• 61
• C N *,
• recombinant plasmid DNA fragment which encode hybrid polypeptide comprising human proinsulin sequence and which is a part of pDIM07 plasmid has the following sequence:
• 1
• ATGGATGAAGACGAGGATGAAGCACGCTTTGTGAACCAGCATCTGTGTGGCAGCCACCTG
• 1
• M D E D E D E A R F V N Q H L C G S H L
• 61
• GTGGAAGCGCTGTATTTAGTGTGCGGCGAGCGTGGCTTCTTTTATACCCCGAAAACCAAA
• 21
• V E A L Y L V C G E R G F F Y T P K T K
• 121
• CGTGGCATTGTGGAACAGTGTTGCACCAGTATTTGTAGCCTGTATCAGCTGGAAAATTAC
• 41
• R G I V E Q C C T S I C S L Y Q L E N Y
• 181
• TGCAACTAA
• 61
• C N *.