Tandem Affinity Purification of SBP-CBP-tagged Type Three Secretion System Effectors

TY - JOUR

T1 - Tandem Affinity Purification of SBP-CBP-tagged Type Three Secretion System Effectors

AU - Berneking, Laura

AU - Schnapp, Marie

AU - Nauth, Theresa

AU - Hentschke, Moritz

N1 - Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.

PY - 2019/6/20

Y1 - 2019/6/20

N2 - Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by co-immunoprecipitation. The tandem affinity purification (TAP) method allows an unbiased screen of potential interaction partners of bacterial effectors in a physiological approach: target cells can be infected with a bacterial strain harboring the TAP-tagged bacterial effector protein which is translocated in the host similar as under physiological infection conditions. No transfection and overexpression of the bacterial protein in the eukaryotic host are needed. Therefore, also host target cells not easy to transfect can be analyzed by this method. Moreover, the two consecutive affinity tags Calmodulin-Binding-Peptide (CBP) and Streptavidin-Binding-Peptide (SBP) fused to the translocated bacterial protein allow an outstanding clear purification of protein complexes formed between the bacterial protein of interest and host cell proteins with less occurrence of contaminants. Mass spectrometry allows an unbiased identification of interacting eukaryotic proteins.

AB - Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by co-immunoprecipitation. The tandem affinity purification (TAP) method allows an unbiased screen of potential interaction partners of bacterial effectors in a physiological approach: target cells can be infected with a bacterial strain harboring the TAP-tagged bacterial effector protein which is translocated in the host similar as under physiological infection conditions. No transfection and overexpression of the bacterial protein in the eukaryotic host are needed. Therefore, also host target cells not easy to transfect can be analyzed by this method. Moreover, the two consecutive affinity tags Calmodulin-Binding-Peptide (CBP) and Streptavidin-Binding-Peptide (SBP) fused to the translocated bacterial protein allow an outstanding clear purification of protein complexes formed between the bacterial protein of interest and host cell proteins with less occurrence of contaminants. Mass spectrometry allows an unbiased identification of interacting eukaryotic proteins.

U2 - 10.21769/BioProtoc.3277

DO - 10.21769/BioProtoc.3277

M3 - SCORING: Journal article

C2 - 33654794

VL - 9

JO - BIO-PROTOCOL

JF - BIO-PROTOCOL

SN - 2331-8325

IS - 12

M1 - e3277

ER -