Selecting Pichia pastoris recombinant clones for higher secretion of human insulin precursor into the culture supernatant

Abstract

The methylotrophic yeast, Pichia pastoris, is one of the preferred yeast hosts for recombinant protein expression. It has been developed as a potential host to express a high level of recombinant proteins, and to achieve efficient secretion as well as growth to very high cell densities. Previously, we have obtained 19 P. pastoris recombinant clones harboring synthetic insulin precursor (IP) expression cassette integrated into their genomes through homologous recombination. To select P. pastoris recombinant clones which exhibit high levels of protein expression, we conducted secreted expressions of IP protein in shake flasks. The secretion of IP into the culture supernatants was verified by SDS-PAGE. IP protein concentrations were estimated using ImageJ by applying lysozyme as standard. All of the 19 P. pastoris recombinant clones were confirmed to secrete the IP protein into their culture supernatants, and a single protein band with a molecular size of approximately 7 kDa was found in the SDS-PAGE gel. The six highest IP-expressing clones were selected for second screening in shake flasks. We selected three recombinant clones (CL-3, CL-4, and CL-18), which secreted the highest levels of IP proteins compared to the other clones. The secreted IP concentrations estimated by ImageJ for clones CL-3, CL-4, and CL-18 were 1230, 1143, and 1010 mg/L, respectively.