Object's details: Identification of protein partners in Mycobacteria using a single step affinity purification method

Provider:Repozytorium Instytutu Biochemii i Biofizyki Polskiej Akademii Nauk

Description

Title:
Identification of protein partners in Mycobacteria using a single step affinity purification method
Creator:
Plocinski, Przemyslaw;
Laubitz, Daniel;
Cysewski, Dominik;
Stodus, Krystian;
Kowalska, Katarzyna;
Dziembowski, Andrzej
Description:
Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spreading and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method is attractively applicable to both medium- and highthroughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. From four tested epitopes, FLAG, eGFP, Protein A, and hemagglutinin, the eGFP tag was found most useful based on easily monitored expression and as a simultaneous tool for sub-cellular localization studies. It presents a relatively low background with cost effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as a bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells, treated with UV light were used to co-purify UvrA interaction partners.
Object availability:
free access
Date:
2014
Type:
Article;
NonPeerReviewed
Language:
English
Format:
application/pdf
Relation: http://eprints.ibb.waw.pl/619 ;http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0091380
Subject:
QH301 Biology
Identifier: http://eprints.ibb.waw.pl/619/1/PlosOne_manuscript.pdf ;
Plocinski, Przemyslaw and Laubitz, Daniel and Cysewski, Dominik and Stodus, Krystian and Kowalska, Katarzyna and Dziembowski, Andrzej (2014) Identification of protein partners in Mycobacteria using a single step affinity purification method. PlosOne, 9 (3). e91380.
Data provider:
Repozytorium Instytutu Biochemii i Biofizyki Polskiej Akademii Nauk

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Object's details: Identification of protein partners in Mycobacteria using a single step affinity purification method

Description

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