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.
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September 1996
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Research Article|
September 01 1996
In vivo and in vitro folding of a recombinant metalloenzyme, phosphomannose isomerase
Amanda E. I. PROUDFOOT;
Amanda E. I. PROUDFOOT
†
1Glaxo Institute for Molecular Biology, 1228 Plan-les-Ouates, Geneva, Switzerland
†To whom correspondence and reprint requests should be sent.
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Laurence GOFFIN;
Laurence GOFFIN
*
1Glaxo Institute for Molecular Biology, 1228 Plan-les-Ouates, Geneva, Switzerland
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Mark A PAYTON;
Mark A PAYTON
1Glaxo Institute for Molecular Biology, 1228 Plan-les-Ouates, Geneva, Switzerland
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Timothy N. C. WELLS;
Timothy N. C. WELLS
1Glaxo Institute for Molecular Biology, 1228 Plan-les-Ouates, Geneva, Switzerland
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Alain R BERNARD
Alain R BERNARD
1Glaxo Institute for Molecular Biology, 1228 Plan-les-Ouates, Geneva, Switzerland
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Publisher: Portland Press Ltd
Received:
January 02 1996
Revision Received:
April 03 1996
Accepted:
April 29 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 318 (2): 437–442.
Article history
Received:
January 02 1996
Revision Received:
April 03 1996
Accepted:
April 29 1996
Citation
Amanda E. I. PROUDFOOT, Laurence GOFFIN, Mark A PAYTON, Timothy N. C. WELLS, Alain R BERNARD; In vivo and in vitro folding of a recombinant metalloenzyme, phosphomannose isomerase. Biochem J 1 September 1996; 318 (2): 437–442. doi: https://doi.org/10.1042/bj3180437
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