Trehalose-6-phosphate synthase 1 from Metarhizium anisopliae: clone, expression and properties of the recombinant.
Trehalose, an important component in fungal spores, is a disaccharide which protects against several environmental stresses, such as heat, desiccation, salt. Trehalose-6-phosphate synthase 1 (TPS1) is a subunit of trehalose synthase complex in fungi; it plays a key role in the biosynthesis of trehalose. In this study, a full-length cDNA from Metarhizium anisopliae encoding TPS1 (designated as MaTPS1) was isolated. The MaTPS1 cDNA is composed of 1836 nucleotides encoding a protein of 517 amino acids with a molecular mass of 58 kDa. The amino acid sequence has a relatively high homology with the TPS1s in several other filamentous fungi. Southern blot analysis showed that MaTPS1 gene occurs as a single copy in the M. anisopliae genome. And MaTPS1 was cloned into Pichia pastoris KM71 and secretively expressed with a histamine tag to facilitate a rapid purification of recombinant MaTPS1 (designated reTPS1). The properties of reTPS1 were examined. The K(m) value of reTPS1 for UDP-glucose and glucose-6-phosphate was 9.6 mM and 3.9 mM, respectively, and the optimal pH and temperature were about 6.5 and 40 degrees C. The enzyme was highly specific to glucose-6-phosphate for glucosyl acceptor, and its activity decreased rapidly as the concentrations of phosphate increased. The expression system will provide sufficient amounts of reTPS1 for future structural characterization of the protein and use for further investigation of MaTPS1's function.
Cai
Zejun
Z
Genetic Engineering Research Center, Chongqing University, Chongqing Engineering Research Center for Fungal Insecticides and Key Lab of Functional Gene and Regulation Technologies under Chongqing Municipal Education Commission, Chongqing 400030, PR China.
Peng
Guoxiong
G
Cao
Yueqing
Y
Liu
Yinchun
Y
Jin
Kai
K
Xia
Yuxian
Y
D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D052981:Metarhizium / Q000201:enzymology* / Q000235:genetics*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase