The main goal of this current study was the cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand using two sets of specific primers which contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, and then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E. coli DH5α competent cells. pPic9k –hpi, which was propagated into the positive transformed E. coli cells, was isolated from cells, linearized by restriction enzyme SalI and then transformed into P. pastoris GS115 using electroporation method. Genomic DNA of His+ transformed cell was extracted and used as a template for PCR analysis. The results show that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformed clones on the anticipated band at 330 bp, which correspond to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformed cells, tricine–SDS gel electrophoresis and Western blotting analysis were conducted. The results show a successful expression of recombinant protein, which was detected by the presence of a single major band with about 5.8 KDa on the gel.

Keywords: Recombinant human insulin, Pichia pastoris, cloning, tricine SDS- page.