Selection of STOP-free sequences from random mutagenesis for `loss of interaction' two-hybrid studies

DC FieldValueLanguage
dc.contributor.authorLickfeld, Manuela
dc.contributor.authorSchmitz, Hans-Peter
dc.date.accessioned2021-12-23T16:01:36Z-
dc.date.available2021-12-23T16:01:36Z-
dc.date.issued2011
dc.identifier.issn0749503X
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/5058-
dc.description.abstractThe investigation of protein-protein interactions is an essential part of biological research. To obtain a deeper insight into regulatory protein networks, the identification of the components, domains and especially single residues that are involved in these interactions is helpful. A widespread and attractive genetic tool for investigation of protein-protein interactions is the yeast two-hybrid system. This method enables large-scale screens and its application is cheap and relatively simple. For identification of the amino acids in a protein sequence that are essential for interaction with a specific partner, yeast two-hybrid assays can be combined with random mutagenesis of the sequence of interest. A common problem with such an experiment is the generation of stop codons within the mutagenized fragments, leading to the isolation of many false positives when screening for loss of interaction using the two-hybrid method. To overcome this problem, we modified the yeast two-hybrid system to allow selection for sequences without stop codons. To achieve this, we fused the ScURA3 marker-gene in frame to the mutagenized fragments. We show here that this marker is fully functional when fused to a two-hybrid construct with a nuclear localization signal, such as a Gal4 activation domain and a prey protein, thus allowing selection of stop-free sequences on media without uracil. Using the Rho-binding domain from a Bni1-like formin and different Rho-type GTPases from Ashbya gossypii as examples, we further show that our system can be used to screen large numbers of transformants for loss of protein-protein interactions in combination with random mutagenesis. Copyright (C) 2011 John Wiley & Sons, Ltd.
dc.language.isoen
dc.publisherWILEY
dc.relation.ispartofYEAST
dc.subjectAshbya gossypii
dc.subjectBiochemistry & Molecular Biology
dc.subjectBiotechnology & Applied Microbiology
dc.subjectESCHERICHIA-COLI
dc.subjectformin
dc.subjectinteraction
dc.subjectMicrobiology
dc.subjectMycology
dc.subjectPCR
dc.subjectPROTEIN INTERACTIONS
dc.subjectrandom mutagenesis
dc.subjectRho-type GTPase
dc.subjecttwo-hybrid
dc.titleSelection of STOP-free sequences from random mutagenesis for `loss of interaction' two-hybrid studies
dc.typejournal article
dc.identifier.doi10.1002/yea.1856
dc.identifier.isiISI:000293226900003
dc.description.volume28
dc.description.issue7
dc.description.startpage535
dc.description.endpage545
dc.contributor.orcid0000-0001-5449-4593
dc.identifier.eissn10970061
dc.publisher.place111 RIVER ST, HOBOKEN 07030-5774, NJ USA
dcterms.isPartOf.abbreviationYeast
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptidfb05-
crisitem.author.orcid0000-0001-5449-4593-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.netidScHa130-
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