Objective, still production of human recombinant insulin is big target for developing countries although
it produced recombinant since 1982. Material and Methods: Human pro-insulin gene codons optimization with aid
of a web based software called OPTIMIZER, where codons that rarely used by E.coli were replaced by abundant
codons, without any changing in the amino acid composition. Pro-insulin long overlap oligos were assembly and
amplified through two successive PCR reactions using different program and polymerase enzyme. The assembly
step called Polymerase Cycling Assembly (PCA), where the second step was ordinary PCR reaction for the gene
amplification. Results: The yielded synthetic gene was cloned into pCR2.1-TOPO cloning vector and transformed
into TOP10F` competent E.coli. The correct sequenced cloned gene was subsequently digested and ligated into
pET101/D/TOPO expression vector followed by transformation into BL21star (DE3) cells. The optimized strain has
been cultivated in shack flask up to 0.6 OD before induction with IPTG. The pro-insulin expression yield and its
characterization will be presented. Conclusion: to built target gene from synthetic overlap oligos, the optimized
primers and cloning, expression vectors were harnessed and presented. |
