Chemical-enzymatic synthesis and cloning of DNA, coding the signal for secretion of proteins in gram-negative bacteria |
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Authors: | V N Dobrynin S V Kalinichenko M R Farkhutdinov S A Filippov V G Korobko |
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Abstract: | Chemical-enzymatic synthesis of an artificial gene encoding leader peptide and 22 N-terminal amino acids of mature carboxypeptidase G2 from Pseudomonas sp. followed by enterokinase signal sequence (Asp4Lys) has been accomplished. The resulted DNA was fused with semi-synthetic gene coding for polypeptide 4-157 of mature human tumour necrosis factor (TNF) and then placed under control of early promoters of T7 bacteriophage. The expression products of the construct obtained was analysed using anti-TNF anti-serum. In E.coli leader peptide was cleaved off during translocation through inner membrane and the resultant product was found in membrane fraction. |
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