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Integrated Tandem Affinity Protein Purification Using the Polyhistidine plus Extra 4 Amino Acids (HiP4) Tag System

ABSTRACT: Interactome analysis of HBx using a novel tag system (HiP4-tag system).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Akihide Ryo

PROVIDER: PXD036256 | JPOST Repository | Tue Feb 28 00:00:00 GMT 2023

REPOSITORIES: jPOST

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			Action	DRS
	His_Only_1.mgf	Mgf
	His_Only_1.raw	Raw
	His_Only_2.mgf	Mgf
	His_Only_2.raw	Raw
	His_Only_3.mgf	Mgf

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Publications

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system.

Ino Yoko Y Yamaoka Yutaro Y Tanaka Kiho K Miyakawa Kei K Nishi Mayuko M Hatayama Yasuyoshi Y Kimura Hirokazu H Kimura Yayoi Y Ryo Akihide A

Proteomics 20230303 11

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed "HiP4" (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this s ...[more]

PMID: 36807525

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Integrated Tandem Affinity Protein Purification Using the Polyhistidine plus Extra 4 Amino Acids (HiP4) Tag System２

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SnAvi--a new tandem tag for high-affinity protein-complex purification.

Project description:Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins.

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Novel affinity tag system using structurally defined antibody-tag interaction: application to single-step protein purification.

Project description:Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody-peptide interaction, opening opportunities for further improvements/modifications.

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Chromatin tandem affinity purification sequencing.

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Project description: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.

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A novel inducible retroviral expression system for StrepHA-tandem affinity purification mass-spectrometry-based proteomics

Project description:Tandem-affinity purification mass spectrometry using the streptavidin-hemagglutinin (SH)-tag has been successfully employed to map signaling networks in several large-scale studies. However its application has so far been restricted to the small number of Flp/FRT-recombination competent cell lines. We present pRSHIC, a novel retroviral, doxycycline-inducible Tet-On vector system suitable for expression of SH-tagged target proteins in a wide range of human and mouse cell systems. The additional feature of concomitant reporter fluorophore expression makes pRSHIC a valuable tool for a diverse set of phenotypic analyses beyond TAP-MS experiments. The dataset demonstrates the application of pRSHIC for TAP-MS analysis of two showcase bait proteins involved in cancer cell proliferation as well as cell death induction and identified novel high- confidence interacting proteins with possible pharmacological intervention potential.

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