In vivo and in vitro folding of a recombinant metalloenzyme, phosphomannose isomerase.
Phosphomannose isomerase (PMI) catalyses the interconversion of mannose 6-phosphate and fructose 6-phosphate in prokaryotic and eukaryotic cells. The enzyme is a metalloenzyme which contains 1 mol of zinc per mol of enzyme. Heterologous expression of the cDNA coding for the Candida albicans enzyme in the prokaryotic host Escherichia coli results in an expression level of up to 30% of total E. coli protein. Ten percent of recombinant PMI is expressed in the soluble fraction and 90% in inclusion bodies. Inclusion of a high level of zinc in the fermentation medium resulted in a fourfold increase in soluble protein. Co-expression of the bacterial chaperones, GroES and GroEL, resulted in a proportional twofold increase in soluble PMI while causing an overall decrease in the PMI expression level. Folding denatured PMI in vitro required reductant and zinc ions. The yield of renatured protein was increased by folding in the presence of GroEL and DnaK in an ATP-independent manner. The refolding yield of denatured soluble enzyme from a guanidine solution was threefold higher than that of folding monomerized inclusion body protein solubilized in guanidine hydrochloride. This suggests that a proportion of recombinant protein expressed in E.coli inclusion bodies may be irreversibly denatured.
Proudfoot
A E
AE
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Goffin
L
L
Payton
M A
MA
Wells
T N
TN
Bernard
A R
AR
D002176:Candida albicans / Q000201:enzymology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008359:Mannose-6-Phosphate Isomerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008667:Metalloproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D006146:Guanidines; D008667:Metalloproteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D008359:Mannose-6-Phosphate Isomerase; D015032:Zinc; D019791:Guanidine; D004229:Dithiothreitol