Abstract
Yeast two-hybrid system combined with the gateway technology will greatly facilitate the cloning of interested DNA fragment into yeast two-hybrid vectors and therefore increase the efficiency of yeast two-hybrid analysis. In this study, we constructed a pair of Gateway-compatible yeast two-hybrid vectors pBTM116GW and pVP16GW by introducing the gateway cassette (attR1-Cmr-ccdB-attR2) into the multiple cloning sites (MCS) of the previously described vectors pBTM116SS and pVP16S1, respectively. The applicability of newly generated vectors was tested by assaying the interaction between the kinase domain XA21K of rice (Oryza sativa) receptor like kinase XA21 and its interactor XB3. Since both Xa21K and Xb3were cloned into a Gateway entry vector and then subcloned into pBTM116GW and pVP16GW by in vitro recombination with high efficiency, respectively, it demonstrated that the newly constructed gateway-compatible two-hybrid vectors will be useful in analysis of protein interactions in a high throughput way. Key words: Yeast two-hybrid, gateway cloning technology, protein interaction
Highlights
Industrial processes such as paper bleaching are usually operated at elevated temperatures
We report a mutant of a family 11 xylanase (Xyn-CDBFV) from Neocallimastix patriciarum with a mutation (D57N) in the active center could be further stabilized by the substrate
Potential hydrogen bonding interactions predicted by molecular docking between the substrate molecule and residues N57, E202 of the mutant are probably responsible for the stabilizing effect
Summary
Industrial processes such as paper bleaching are usually operated at elevated temperatures. Ideal industrial enzymes such as xylanase should be highly active at high temperatures. It is very interesting to engineer thermophilic xylanases, which have been created from mesophilic enzymes by site-directed mutagenesis and directed evolution (Dumon et al, 2008; Jeong et al, 2007; Miyazaki et al, 2006; Ruller et al, 2008; Stephens et al, 2007). Substrate molecules have long been known to be able to improve the enzyme thermostability by stabilizing the active site (Vieille and Zeikus, 2001). It was obvious that a good understanding of the molecular basis for such effect may lead to new rules for rational design and engineering of thermophilic xylanases
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