title: Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers.
abstract: Overexpression of recombinant N-acetyl-D-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08±0.02 U/mg, was similar to that of the native protein, 2.13±0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41±10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32±5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins.
authors: Department of Chemical Engineering, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan.
authors: Shih-Chin
authors: SC
authors: Lu
authors: Sung-Chyr
authors: SC
authors: Lin
mesh_terms: D000818:Animals; D057005:Bioengineering; D002021:Buffers; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D002479:Inclusion Bodies / Q000201:enzymology; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013552:Swine
chemical_list: D002021:Buffers; D002352:Carrier Proteins; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase