Full Length Research Paper

Construction of gateway-compatible yeast two-hybrid vectors for high throughput analysis of protein interaction

Tingheng Zhu¹, Weixia Wang²*, Hann-lin Wong³, Xiao Yang¹, Kun Wang¹ and Zhifeng Cui¹*

¹College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China.

²China National Rice Research Institute, Hangzhou 310006, China.

³Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma 630-0101, Japan.

*Corresponding authors. E-mail: wxiawang@126.com. Tel: 86-571-63370348 or 86-571-63370359. zfcui@zjut.edu.cn. Tel: 86-571-88320741.

Abbreviations: MCS, Multiple cloning sites; SD, synthetic medium; DB, DNA-binding domain; LB, Luria-Bertani; AD, activation domain; PCR, polymerase chain reaction.

Accepted 14 January, 2010

   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 Xb3 were 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.