Expression, purification, and crystallization of a novel galactose mutarotase from Thermoanaerobacter tengcongensis.
- Author:
Lan WU
1
;
Zhong QIAN
;
Jun FU
;
Shi-ying MIAO
;
Lin-fang WANG
Author Information
- Publication Type:Journal Article
- MeSH: Bacterial Proteins; biosynthesis; chemistry; isolation & purification; Carbohydrate Epimerases; biosynthesis; chemistry; isolation & purification; Cloning, Molecular; Crystallization; Escherichia coli; genetics; metabolism; Genetic Vectors; Thermoanaerobacter; enzymology; genetics; Transformation, Bacterial
- From: Acta Academiae Medicinae Sinicae 2009;31(6):696-701
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.
METHODSThe tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200).
RESULTThe purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained.
CONCLUSIONSWe successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.
