Improved Refolding of an Immobilized Fusion Protein
Improved Refolding of an Immobilized Fusion Protein
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DOI：
10.1038/nbt0396-329
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发表时间：
1996-03
影响因子：
46.9
通讯作者：
Günter Stempfer;B. Höll-Neugebauer;R. Rudolph
中科院分区：
文献类型：
--
作者：
Günter Stempfer;B. Höll-Neugebauer;R. Rudolph
Fusion proteins of monomeric α-glucosidase fromSaccharomyces cerevisiaecontaining N- or C-terminal hexa-arginine peptides were expressed in the cytosol ofEscherichia coliin 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 α-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 μg/ml). Thus, folding of the immobilized α-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.
