Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica ›› 2009, Vol. 31 ›› Issue (6): 696-701.doi: 10.3881/j.issn.1000-503X.2009.06.008

• Original Articles • Previous Articles     Next Articles

Expression, Purification, and Crystallization of A Novel Galactose Mutarotase from Thermoanaerobacter Tengcongensis

WU Lan1; QIAN Zhong2; FU Jun1;MIAO Shi-ying1;WANG Lin-fang1   

  1. 1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China
    2Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, China
  • Received:2009-09-27 Revised:1900-01-01 Online:2009-12-30 Published:2009-12-30
  • Contact: WANG Lin-fang

Abstract: ABSTRACT: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 β-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione SepharoseTM4B affinity, ion chromatography (Resource Q 6mL), 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 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.

Key words: tdermoanaerobacter tencconcensis, tte1925, calactose mutarotase, prokaryotic expression, protein purification, crystallization