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. 1996 Sep 1;318(Pt 2):437–442. doi: 10.1042/bj3180437

In vivo and in vitro folding of a recombinant metalloenzyme, phosphomannose isomerase.

A E Proudfoot 1, L Goffin 1, M A Payton 1, T N Wells 1, A R Bernard 1
PMCID: PMC1217640  PMID: 8809030

Abstract

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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Selected References

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  1. Bernard A. R., Wells T. N., Cleasby A., Borlat F., Payton M. A., Proudfoot A. E. Selenomethionine labelling of phosphomannose isomerase changes its kinetic properties. Eur J Biochem. 1995 May 15;230(1):111–118. doi: 10.1111/j.1432-1033.1995.0111i.x. [DOI] [PubMed] [Google Scholar]
  2. Caspers P., Stieger M., Burn P. Overproduction of bacterial chaperones improves the solubility of recombinant protein tyrosine kinases in Escherichia coli. Cell Mol Biol (Noisy-le-grand) 1994 Jul;40(5):635–644. [PubMed] [Google Scholar]
  3. Coulin F., Magnenat E., Proudfoot A. E., Payton M. A., Scully P., Wells T. N. Identification of Cys-150 in the active site of phosphomannose isomerase from Candida albicans. Biochemistry. 1993 Dec 28;32(51):14139–14144. doi: 10.1021/bi00214a010. [DOI] [PubMed] [Google Scholar]
  4. Dale G. E., Schönfeld H. J., Langen H., Stieger M. Increased solubility of trimethoprim-resistant type S1 DHFR from Staphylococcus aureus in Escherichia coli cells overproducing the chaperonins GroEL and GroES. Protein Eng. 1994 Jul;7(7):925–931. doi: 10.1093/protein/7.7.925. [DOI] [PubMed] [Google Scholar]
  5. Fisher M. T. Promotion of the in vitro renaturation of dodecameric glutamine synthetase from Escherichia coli in the presence of GroEL (chaperonin-60) and ATP. Biochemistry. 1992 Apr 28;31(16):3955–3963. doi: 10.1021/bi00131a010. [DOI] [PubMed] [Google Scholar]
  6. Goloubinoff P., Christeller J. T., Gatenby A. A., Lorimer G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature. 1989 Dec 21;342(6252):884–889. doi: 10.1038/342884a0. [DOI] [PubMed] [Google Scholar]
  7. Gracy R. W., Noltmann E. A. Studies on phosphomannose isomerase. I. Isolation, homogeneity measurements, and determination of some physical properties. J Biol Chem. 1968 Jun 10;243(11):3161–3168. [PubMed] [Google Scholar]
  8. Gracy R. W., Noltmann E. A. Studies on phosphomannose isomerase. II. Characterization as a zinc metalloenzyme. J Biol Chem. 1968 Aug 10;243(15):4109–4116. [PubMed] [Google Scholar]
  9. Hartl F. U., Martin J., Neupert W. Protein folding in the cell: the role of molecular chaperones Hsp70 and Hsp60. Annu Rev Biophys Biomol Struct. 1992;21:293–322. doi: 10.1146/annurev.bb.21.060192.001453. [DOI] [PubMed] [Google Scholar]
  10. Landry S. J., Gierasch L. M. The chaperonin GroEL binds a polypeptide in an alpha-helical conformation. Biochemistry. 1991 Jul 30;30(30):7359–7362. doi: 10.1021/bi00244a001. [DOI] [PubMed] [Google Scholar]
  11. Lee S. C., Olins P. O. Effect of overproduction of heat shock chaperones GroESL and DnaK on human procollagenase production in Escherichia coli. J Biol Chem. 1992 Feb 15;267(5):2849–2852. [PubMed] [Google Scholar]
  12. Martin J., Langer T., Boteva R., Schramel A., Horwich A. L., Hartl F. U. Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate. Nature. 1991 Jul 4;352(6330):36–42. doi: 10.1038/352036a0. [DOI] [PubMed] [Google Scholar]
  13. Martin J., Mayhew M., Langer T., Hartl F. U. The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding. Nature. 1993 Nov 18;366(6452):228–233. doi: 10.1038/366228a0. [DOI] [PubMed] [Google Scholar]
  14. McCarty J. S., Buchberger A., Reinstein J., Bukau B. The role of ATP in the functional cycle of the DnaK chaperone system. J Mol Biol. 1995 May 26;249(1):126–137. doi: 10.1006/jmbi.1995.0284. [DOI] [PubMed] [Google Scholar]
  15. Mendoza J. A., Rogers E., Lorimer G. H., Horowitz P. M. Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J Biol Chem. 1991 Jul 15;266(20):13044–13049. [PubMed] [Google Scholar]
  16. Payton M. A., Rheinnecker M., Klig L. S., DeTiani M., Bowden E. A novel Saccharomyces cerevisiae secretory mutant possesses a thermolabile phosphomannose isomerase. J Bacteriol. 1991 Mar;173(6):2006–2010. doi: 10.1128/jb.173.6.2006-2010.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Perkins T. T., Smith D. E., Chu S. Direct observation of tube-like motion of a single polymer chain. Science. 1994 May 6;264(5160):819–822. doi: 10.1126/science.8171335. [DOI] [PubMed] [Google Scholar]
  18. Proudfoot A. E., Fattah D., Kawashima E. H., Bernard A., Wingfield P. T. Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli. Biochem J. 1990 Sep 1;270(2):357–361. doi: 10.1042/bj2700357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Proudfoot A. E., Turcatti G., Wells T. N., Payton M. A., Smith D. J. Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase. Eur J Biochem. 1994 Jan 15;219(1-2):415–423. doi: 10.1111/j.1432-1033.1994.tb19954.x. [DOI] [PubMed] [Google Scholar]
  20. Smith D. J., Proudfoot A. E., Detiani M., Wells T. N., Payton M. A. Cloning and heterologous expression of the Candida albicans gene PMI 1 encoding phosphomannose isomerase. Yeast. 1995 Apr 15;11(4):301–310. doi: 10.1002/yea.320110402. [DOI] [PubMed] [Google Scholar]
  21. Smith D. J., Proudfoot A., Friedli L., Klig L. S., Paravicini G., Payton M. A. PMI40, an intron-containing gene required for early steps in yeast mannosylation. Mol Cell Biol. 1992 Jul;12(7):2924–2930. doi: 10.1128/mcb.12.7.2924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wells T. N., Coulin F., Payton M. A., Proudfoot A. E. Phosphomannose isomerase from Saccharomyces cerevisiae contains two inhibitory metal ion binding sites. Biochemistry. 1993 Feb 9;32(5):1294–1301. doi: 10.1021/bi00056a014. [DOI] [PubMed] [Google Scholar]

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