1. Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product
Y Wang, Z H Liang, Y S Zhang, S Y Yao, Y G Xu, Y H Tang, S Q Zhu, D F Cui, Y M Feng Biotechnol Bioeng. 2001 Apr 5;73(1):74-9. doi: 10.1002/1097-0290(20010405)73:13.0.co;2-v.
The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form.
2. Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter
Diego Gabriel Noseda, Matías Nicolás Recúpero, Martín Blasco, Gastón Ezequiel Ortiz, Miguel Angel Galvagno Protein Expr Purif. 2013 Dec;92(2):235-44. doi: 10.1016/j.pep.2013.08.018. Epub 2013 Oct 16.
The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.
3. Secretory expression of insulin precursor in pichia pastoris and simple procedure for producing recombinant human insulin
T Xie, Q Liu, F Xie, H Liu, Y Zhang Prep Biochem Biotechnol. 2008;38(3):308-17. doi: 10.1080/10826060802165147.
In this work, Pichia pastoris was applied to produce human insulin by a simple procedure. The synthesized insulin precursor (ILP) gene was inserted into pPIC9K to obtain secretary expression plasmid pPIC9K/ILP. Pichia pastoris GS115 was transformed by pPIC9K/ILP and the high expresser was screened. In a 16 L fermentor, the insulin precursor production was 3.6 g/L. Insulin precursor, purified by one-step chromatography, was converted into human insulin by transpeptidation. The yield of the processing procedure from insulin precursor to insulin reached up to 70%. In vivo assay showed that the biological activity of the produced recombinant human insulin was 28.8 U/mg.
