title: Tools for fungal proteomics: multifunctional neurospora vectors for gene replacement, protein expression and protein purification.
abstract: The completion of genome-sequencing projects for a number of fungi set the stage for detailed investigations of proteins. We report the generation of versatile expression vectors for detection and isolation of proteins and protein complexes in the filamentous fungus Neurospora crassa. The vectors, which can be adapted for other fungi, contain C- or N-terminal FLAG, HA, Myc, GFP, or HAT-FLAG epitope tags with a flexible poly-glycine linker and include sequences for targeting to the his-3 locus in Neurospora. To introduce mutations at native loci, we also made a series of knock-in vectors containing epitope tags followed by the selectable marker hph (resulting in hygromycin resistance) flanked by two loxP sites. We adapted the Cre/loxP system for Neurospora, allowing the selectable marker hph to be excised by introduction of Cre recombinase into a strain containing a knock-in cassette. Additionally, a protein purification method was developed on the basis of the HAT-FLAG tandem affinity tag system, which was used to purify HETEROCHROMATIN PROTEIN 1 (HP1) and associated proteins from Neurospora. As expected on the basis of yeast two-hybrid and co-immunoprecipitation (Co-IP) experiments, the Neurospora DNA methyltransferase DIM-2 was found in a complex with HP1. Features of the new vectors allowed for verification of an interaction between HP1 and DIM-2 in vivo by Co-IP assays on proteins expressed either from their native loci or from the his-3 locus.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
authors.forename: Shinji
authors.initials: S
authors.lastname: Honda
authors.forename: Eric U
authors.initials: EU
authors.lastname: Selker
mesh_terms: D002846:Chromatography, Affinity; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000302:isolation & purification; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D047468:Immunoprecipitation; D019426:Integrases; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics*; D010957:Plasmids; D040901:Proteomics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D002868:Chromosomal Proteins, Non-Histone; C484364:DIM2 protein, S cerevisiae; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D000090266:Chromobox Protein Homolog 5; C045073:Cre recombinase; D019426:Integrases