Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology.
Response Surface Methodology (RSM) was used to investigate optimal refolding conditions of Pseudomonas fluorescens lipase which was expressed as inclusion body in E. coli. Three interacting factors, protein concentration, pH and guanidine hydrochloride (GdnHCl) concentration were selected as the variables of RSM. The protein concentration did not affect the refolding yield within the selected range (50-340 micrograms ml-1) at low temperatures of 4 and -15 degrees C, but it was a critical factor at 25 degrees C and refolding yield significantly decreased with increasing protein concentration. The pH and GdnHCl were significant factors in all experimental conditions. But there was no trends of optimal pH depending on temperature and additives, just showing the optimum at neutral pH. Glycerol shifted optimal GdnHCl concentration to higher side, while arginine to lower side. Therefore, it was concluded that glycerol and arginine deprives and supplements GdnHCl, respectively. In this study, RSM made it possible to investigate successfully the optimal conditions of in vitro refolding and to elucidate interactions between refolding factors with a minimum number of experiments. Under the optimal condition determined by RSM, approximately 90% refolding yield was obtained and it was a 30% increase over the conventional method.
Ahn
J H
JH
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Lee
Y P
YP
Rhee
J S
JS
D001120:Arginine / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005990:Glycerol / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000201:enzymology / Q000235:genetics; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D006146:Guanidines; D011994:Recombinant Proteins; D001120:Arginine; D008049:Lipase; D019791:Guanidine; D005990:Glycerol