Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.
Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC.
Key Laboratory of Synthetic and Natural Functional Molecular Chemistry of Ministry of Education, Institute of Modern Separation Science, Shaanxi Key laboratory of Modern Separation Science, Northwest University, Xi'an, 710068, China.
Jie
J
Yuan
Huifang
H
Zhou
Yicong
Y
Yang
Weimin
W
Li
Yi
Y
Wan
Lili
L
Wang
D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D011384:Proinsulin
bio-separations; protein refolding recombinant human proinsulin; protein-folding liquid chromatography (PFLC)