Display Settings:

Format

Send to:

Choose Destination
Mol Biochem Parasitol. 2011 Jul-Aug;178(1-2):56-9. doi: 10.1016/j.molbiopara.2011.04.005. Epub 2011 Apr 20.

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

Author information

  • 1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.

Abstract

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 portions of P. falciparum genes to be expressed and screened in the yeast two-hybrid assay. The PCR-based method is less technically challenging and facilitates fine-scale mapping of protein interaction domains. Both approaches revealed a putative interaction between PfMyb2 (PF10_0327) and PFC0365w. We developed new plasmids to express the proteins in wheat germ extracts and confirmed the interaction in both the split-luciferase assay and in co-purification experiments with glutathione-S-transferase and HA-tagged proteins. The combination of improved yeast two-hybrid screening approaches and convenient systems to validate interactions enhances the utility of yeast two-hybrid assays for P. falciparum.

Copyright © 2011 Elsevier B.V. All rights reserved.

PMID:
21530591
[PubMed - indexed for MEDLINE]
PMCID:
PMC3120127
Free PMC Article

Images from this publication.See all images (2)Free text

Fig. 1
Fig. 2
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science Icon for PubMed Central
    Loading ...
    Write to the Help Desk