The main goal of the current study was 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, by using two sets of specific primers contain EcoR1 and Notl restriction sites.
The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, 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 in the positive transformant E. coli
cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115
using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR
analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected
His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of
the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western
blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the
presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human
insulin with the theoretical molecular weight (5.8 KDa).
Selecting Pichia pastoris recombinant clones for higher secretion of human insulin precursor into the culture supernatant
