Open AccessCapture and intermediate purification of human insulin precursor from Pichia pastoris culture using cation exchange chromatography
Author(s) -
Eko Wahyu Putro,
Dini Nurdiani,
Hariyatun,
Ning Wikan Utami,
Wien Kusharyoto
Publication year - 2021
Publication title -
iop conference series. earth and environmental science
Language(s) - English
Resource type - Journals
eISSN - 1755-1307
pISSN - 1755-1315
DOI - 10.1088/1755-1315/762/1/012028
Subject(s) - chromatography , elution , pichia pastoris , chemistry , centrifugation , size exclusion chromatography , high performance liquid chromatography , absorbance , ion exchange , lysozyme , protein purification , ion chromatography , recombinant dna , biochemistry , ion , organic chemistry , gene , enzyme
Purification has an important role in obtaining protein with a high degree of purity, particularly for human therapeutic purposes. Purification of pharmaceutical proteins requires several unit operations, involving chromatographic separation techniques. To increase purification efficiency and shorten process development, it is necessary to examine the chromatography system in performing a capture and intermediate purification in a single step. We use one of the best Pichia pastoris clones obtained from previous studies to produce a human insulin precursor (HIP). To capture and purify HIP from the culture, we clarified the cells through centrifugation and filtration. The supernatant was then loaded into a cation exchange column. Purification was carried on by two-step elution and monitored based on UV absorbance. Effects of loading concentration, flow rate, and pH of samples were evaluated. Fractions of elution were collected and verified by SDS-PAGE. Concentrations of HIP protein were quantified by using ImageJ by incorporating lysozyme as standard and reversed phase HPLC. Loading concentration and pH of the sample have an impact on the recovery. In this study, the best HIP recovery at ∼ 47% resulted from purification with 10% volume of loading concentration and 500 ml loading volume.