One step purification of biological active human interleukin-2 protein produced in yeast (Pichia Pastoris)

Abstract

Pharmacological importance of recombinant human interleukin-2 protein has increased the demand to establish effective, reliable and cost effective chromatography method for its production and purification on large scale. One step mimetic ligand affinity chromatography method for purification of mutated human recombinant interleukin-2 (mrhIL-2), from Pichia Pastoris is described with higher yield then reported before. The mrhIL-2 was expressed extracellularly under methanol inducible AOX1 promoter of P. pastoris. Extracellular expression of mrhIL-2 in the culture supernatant was ~210 mg/L. Cell free culture supernatant containing mrhIL-2 protein was concentrated and buffer exchanged by diafiltration by tangential flow filtration system. Different mimetic legends column from sigma were screened for efficient binding of mrhIL-2 in culture supernatant. Maximum binding was observed with Mimetic Blue SA P6XL and least with Mimetic Green 1 A6XL. One step Dye ligand affinity chromatography method was developed by using Mimetic Blue SA P6XL ligand for high level purification of mutant of interleukin-2. Final yield of purified protein was 115 mg/L and purity of 97%. The interleukin-2 protein prepared by this protocol was found to be monomeric based on SDS-PAGE, western blot and HPLC analysis. Purified protein was biological active as checked by cell proliferation assay.

Key words: Interleukin-2, Pichia pastoris, Dye ligand affinity chromatography.