Chaperone-assisted column refolding of gloshedobin with the use of refolding cocktail.
Gloshedobin, a recently isolated thrombin-like enzyme from the snake venom of Gloydius shedaoensis, is expressed mainly in the form of inclusion bodies (IBs) in Escherichia coli due to its cysteine-rich nature. Following extraction and solubilization of the IBs, one-step immobilized metal affinity chromatography purification produces highly purified (>99%) denatured-solubilized gloshedobin ready to enter the subsequent refolding process. However, the traditional dilution or column refolding strategy, based on gradual denaturant removal, is found to be inefficient for the recovery of protein activity. In this study, a new refolding strategy harnessing the ClpB and DnaK/DnaJ/GrpE bichaperone system is demonstrated to be superior to the conventional refolding methods in either batch dilution or column refolding mode. It is noted that the efficacy of bichaperone-mediated column refolding strategy is further highlighted especially when refolding reaction is attempted at a higher protein concentration with the recirculation of the refolding cocktail containing the bichaperone system. This is evidenced by an uncompromised refolding efficiency (ca. 21.4%) achieved at 2000 microg/mL of initial protein concentration, which is comparable to the refolding efficiency (ca. 22.5%) obtained at 20 times lower protein concentration (i.e. 100 microg/mL) in the conventional batch dilution refolding technique. The demonstrated chaperone-assisted column refolding strategy thus provides an effective tool for refolding-recalcitrant proteins whose reactivation is otherwise difficult to achieve.
Nian
Rui
R
Department of Chemical & Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore.
Tan
Lihan
L
Yoo
Ik-Keun
IK
Choe
Woo-Seok
WS
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione; D019803:Glutathione Disulfide; D006360:Heat-Shock Proteins / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013917:Thrombin / Q000235:genetics / Q000378:metabolism; D017819:Viperidae
D003435:Crotalid Venoms; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D013917:Thrombin; D005978:Glutathione; D019803:Glutathione Disulfide