Improved refolding of an immobilized fusion protein.
Fusion proteins of monomeric alpha-glucosidase from Saccharomyces cerevisiae containing N- or C-terminal hexa-arginie peptides were expressed in the cytosol of Escherichia coli in soluble form. The polycationic peptide moieties allow noncovalent binding of the denatured fusion proteins to a polyanionic solid support. Upon removal of the denaturant, refolding of the matrix-bound protein can proceed without perturbation by aggregation. However, nonspecific interactions of the denatured polypeptide, or of folding intermediates, with the matrix cause a drastic decrease in renaturation under suboptimal folding conditions. At low salt concentrations, ionic interactions of the refolding polypeptide with the matrix result in lower yields of renaturation. At higher salt concentrations, renaturation is prevented by hydrophobic interactions with the matrix. Apart from ionic strength, renaturation of the denatured matrix-bound fusion protein must be optimized with respect to pH, temperature, cosolvents, and matrix material used. Under optimum conditions, immobilized alpha-glucosidase can be renatured with a high yield at protein concentrations up to 5 mg/ml, whereas folding of the wild-type enzyme in solution is feasible only at an extremely low protein concentration (15 micrograms/ml). Thus, folding of the immobilized alpha-glucosidase allows an extremely high yield of the renaturated model protein. The technology should be applicable to other proteins that tend to aggregate during refolding.
Stempfer
G
G
Boehringer Mannheim Therapeutics, Penzberg, Germany.
Höll-Neugebauer
B
B
Rudolph
R
R
D001709:Biotechnology; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000235:genetics; D009994:Osmolar Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012965:Sodium Chloride; D013696:Temperature; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics
D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D000520:alpha-Glucosidases