Interactome mapping in malaria parasites: challenges and opportunities.
Nearly two-thirds of the proteins encoded by Plasmodium falciparum, the parasite that causes the most deadly form of malaria, are annotated as "hypothetical." The yeast two-hybrid assay, which requires no prior knowledge about the target protein, has great potential to provide functional information about these uncharacterized proteins. However, P. falciparum yeast two-hybrid screens are hampered by the poor expression of P. falciparum genes in yeast. AU-rich sequences in nascent P. falciparum transcripts resemble the 3' end processing sites in yeast mRNAs, and are prematurely cleaved and polyadenylated. In most cases, these aberrant messages are degraded and yield no protein. To overcome this limitation, we have developed methods to extensively fragment P. falciparum genes. Novel yeast two-hybrid vectors, in which auxotrophic markers are fused to the 3' ends of the cloned inserts, are employed to identify those gene fragments that are expressed in yeast. In this chapter, we provide detailed protocols for fragmenting P. falciparum genes, creating P. falciparum activation domain libraries, and performing P. falciparum yeast two-hybrid screens. Though focused on P. falciparum, the approaches described here are applicable to other organisms and are likely to be especially useful for those with AT-rich genomes, which are also likely to be poorly expressed in yeast.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA.
Douglas J
DJ
LaCount
D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D003850:Deoxyribonuclease I / Q000378:metabolism; D017125:Genes, Protozoan / Q000235:genetics; D008288:Malaria / Q000469:parasitology*; D010957:Plasmids / Q000235:genetics; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D015800:Protozoan Proteins; D004247:DNA; D003850:Deoxyribonuclease I