Improving yeast two-hybrid screening systems.

Yeast Two Hybrid cDNA Screening of Arabidopsis thaliana for SETH4 Protein Interaction

SETH4 coding sequence with 2013 bp is a member of gene family expressed in gametophytic tissues of Arabidopsis thaliana. This fragment was PCR amplified using Kod Hi Fi DNA polymerase enzyme. This fragment was cloned into pGBKT7 bate vector and transformed E. coli DH5? cells containing vector were selected on LB medium containing Kanamycin. Finally, pGBKT7-SETH4 bate was transformed into yeast ...

A combined yeast/bacterial two-hybrid system (YBTH): development and evaluation

Two-hybrid screening is a standard methodology to identify and characterize protein-protein interactions that has become an integral component of many proteomic investigations. The twohybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bact...

Functional analyses and affinity-alteration of receptors and enzymes based on membrane recruitment of yeast guanine nucleotide-binding protein subunit

To investigate fundamental processes conserved in all eukaryotic cells, the budding yeast Saccharomyces cerevisiae has being widely used as a model organism. In particular, the yeast two-hybrid system is a powerful technique for identifying protein–protein interactions and analyzing protein function in living cells. Here, we describe several approaches for investigating and regulating the activ...

A CRISPR-based yeast two-hybrid system for investigating RNA-protein interactions

A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins.

Use of the yeast two-hybrid assay to study Plasmodium falciparum protein-protein interactions is limited by poor expression of P. falciparum genes in yeast and lack of easily implemented assays to confirm the results. We report here two methods to create gene fragments - random fragmentation by partial DNAse I digestion and generation of densely overlapping fragments by PCR - that enable most p...