earticle

영어

Human insulin is generally genetically synthesized by expressing proinsulin in a host cell followed by harvesting proinsulin, treating its with trypsin-carboxyl peptidase B to remove the internal C-polypeptide to result in the active insulin. When the 35-aminoacid C-polypeptide in proinsulin was replaced by a short linker peptide of 9 amino acids, the resulting mini-proinsulin was reported to have an improved folding efficiency and, consequently, to give better insulin production yield. On the other hand, recombinant protein in E. coli normally forms inclusion body which has to be solubilized by strong chaotropic agents. In the case of recombinant proinsulin or mini-proinsulin, this solubilized step can result in large amount of misfold molecules which affects the total yield of insulin. In order to avoid this solubilization step, we designed a gene construct which permits to generate mini-proinsulin under soluble form in E. coli cell. The gene encoding mini-proinsulin (MPI) was amplified by PCR, using synthetic primers basing on the Genbank registered human insulin sequence with accession number NM_000207. The 5’ end of the gene was fused downstream to the gene encoding the heavy chain of human ferritin (FTNH), which has been cloned onto pET28a expression vector. A dipeptide linkage specifically cleaved by trypsin was introduced between FTNH and MPI to facilitate the remove of the former from the fused FTNH-MPI protein. FTNH has been reported to improve the solubility of exogenous proteins in E. coli when its was co-expressed with E. coli DnaK chaperone. Therefore, dnaK gene was amplified from E. coli genome by PCR and cloned onto pET43.1.a expression vector. Recombinant vector was transformed into E. coli BL21 (DE3). Transformant with recombinat vector was selected and used as host to be transformed with recombinant vector carrying ftnh-mpi gene. Successfully selected clone was cultured and induced by 0.5 mM IPTG for 20 hours to expresse the fused FTNH-MPI. Cell homogenate was fractionated by centrifugation and the presence of FTNH-MPI in the cytosolic fraction was successfully confirmed by SDS-PAGE and Western blot. FTNH-MPI was treated with trypsin to remove FTNH from the fused protein and the mini-C peptide from mini-proinsulin. Resulting active insulin was confirmed by ELISA using a specific monoclonal antibody which has been reported to bind to active insulin.