[Expression, purification, and crystallization of a novel galactose mutarotase from Thermoanaerobacter tengcongensis].
OBJECTIVE
To purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.


METHODS
The 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).


RESULTS
The 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.


CONCLUSIONS
We 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.
Wu
Lan
L
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing 100005, China.
Qian
Zhong
Z
Fu
Jun
J
Miao
Shi-ying
SY
Wang
Lin-fang
LF
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D002238:Carbohydrate Epimerases; C459183:galactose mutarotase