scholarly journals Establishment of Proximity-dependent Biotinylation Approaches in Different Plant Model Systems

2019 ◽  
Author(s):  
Deepanksha Arora ◽  
Nikolaj B. Abel ◽  
Chen Liu ◽  
Petra Van Damme ◽  
Klaas Yperman ◽  
...  
Keyword(s):  
Structural Information ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Lotus Japonicus ◽  
Model Systems ◽  
Test Case ◽  
Plant Model ◽  
One Step ◽  
Subsequent Capture ◽  
Biotin Labelling

AbstractProximity-dependent biotin labelling (PDL) uses a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest. This enables covalent biotin labelling of proteins and allows subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report on the use of PDL in plants. Here we present the results of a systematic study applying a variety of PDL approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols which combine Mass Spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both non-biotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes.

scholarly journals MRG proteins are shared by multiple protein complexes with distinct functions

2021 ◽  
Author(s):  
Maeva Devoucoux ◽  
Celine Roques ◽  
Charles Joly-Beauparlant ◽  
Arnaud Droit ◽  
Samer M.I. Hussein ◽  
...  
Keyword(s):  
Dna Repair ◽  
Structural Information ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Point Mutations ◽  
K562 Cells ◽  
Coding Region ◽  
Multiple Protein ◽  
Alternative Mrna Splicing ◽  
Endogenous Proteins

MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain recognizing H3K36me3 in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing and DNA repair by homologous recombination. In order to get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the chromodomain, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase and PALB2/BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR-tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that can specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding chromodomain of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes which is essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.

scholarly journals Cleavable Affinity Purification (Cl-AP): A One-step Procedure to Affinity Purify Protein Complexes

BIO-PROTOCOL ◽  
2020 ◽  
Vol 10 (22) ◽  
Author(s):  
Ammarah Tariq ◽  
Lucy Green ◽  
Christian Soeller ◽  
James Wakefield
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Step Procedure ◽  
One Step

scholarly journals Genetic and Proteomic Analysis of Rna Polymerase Ii-Interacting Complexes Mediator and Integrator in the Ciliated Protozoan Tetrahymena Thermophila

2021 ◽  
Author(s):  
Matthew D. R. Cadorin
Keyword(s):  
Rna Polymerase Ii ◽  
Tetrahymena Thermophila ◽  
Slow Growth ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Model Systems ◽  
Ciliated Protozoan ◽  
Conventional Model ◽  
Colony Pcr ◽  
Independent Functions

In most eukaryotes, the largest subunit of RNAPII, Rpb1, contains a conserved carboxyterminal domain (CTD) containing a canonical structure of heptapeptide repeats. Two protein complexes of interest, Mediator and Integrator, are known to interact with this CTD in all eukaryotic models they have been described in to date. Recently, orthologs of Mediator and Integrator subunits have been identified within the ciliated protozoan Tetrahymena thermophila; one of the few eukaryotic lineages to lack a canonically organized CTD. To begin to characterize putative Mediator and Integrator complexes within T. thermophila, I engineered appropriate macronuclear tagging and knockout cassettes. Although the Tetrahymena MED31 ortholog was unable to rescue the slow growth phenotype of a yeast MED31 knockout, or co-purify with yeast Med8-TAP, I identified subunit Med3 as a member of the Med31 interactome in T. thermophila through tandem affinity purification coupled with mass spectrometry. I also targeted the Tetrahymena INTS6 locus for knockout as determined by colony PCR. If Mediator and Integrator exist in Tetrahymena despite its divergent CTD of Rpb1, perhaps these complexes have CTD-independent functions beyond what can be effectively studied using conventional model systems.

scholarly journals Genetic and Proteomic Analysis of Rna Polymerase Ii-Interacting Complexes Mediator and Integrator in the Ciliated Protozoan Tetrahymena Thermophila

2021 ◽  
Author(s):  
Matthew D. R. Cadorin
Keyword(s):  
Rna Polymerase Ii ◽  
Tetrahymena Thermophila ◽  
Slow Growth ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Model Systems ◽  
Ciliated Protozoan ◽  
Conventional Model ◽  
Colony Pcr ◽  
Independent Functions

In most eukaryotes, the largest subunit of RNAPII, Rpb1, contains a conserved carboxyterminal domain (CTD) containing a canonical structure of heptapeptide repeats. Two protein complexes of interest, Mediator and Integrator, are known to interact with this CTD in all eukaryotic models they have been described in to date. Recently, orthologs of Mediator and Integrator subunits have been identified within the ciliated protozoan Tetrahymena thermophila; one of the few eukaryotic lineages to lack a canonically organized CTD. To begin to characterize putative Mediator and Integrator complexes within T. thermophila, I engineered appropriate macronuclear tagging and knockout cassettes. Although the Tetrahymena MED31 ortholog was unable to rescue the slow growth phenotype of a yeast MED31 knockout, or co-purify with yeast Med8-TAP, I identified subunit Med3 as a member of the Med31 interactome in T. thermophila through tandem affinity purification coupled with mass spectrometry. I also targeted the Tetrahymena INTS6 locus for knockout as determined by colony PCR. If Mediator and Integrator exist in Tetrahymena despite its divergent CTD of Rpb1, perhaps these complexes have CTD-independent functions beyond what can be effectively studied using conventional model systems.

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

1990 ◽  
Vol 63 (03) ◽  
pp. 439-444 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P Walder ◽  
P W Straub
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Terminal Sequence ◽  
Purification Method ◽  
Human Fibrinogen ◽  
Isolation Methods ◽  
One Step ◽  
Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

2020 ◽  
Vol 27 (37) ◽  
pp. 6306-6355 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia Anna Mercurio ◽  
Marilisa Leone
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Protein Interactions ◽  
Modular Architecture ◽  
Post Translational Modifications ◽  
Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

Affinity Tags for Protein Purification

2020 ◽  
Vol 21 (8) ◽  
pp. 821-830
Author(s):  
Vibhor Mishra
Keyword(s):  
Protein Purification ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein Domain ◽  
Affinity Tag ◽  
Small Peptides ◽  
Affinity Tags ◽  
C Terminus ◽  
Solubility Enhancers ◽  
As Solubility

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

scholarly journals Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes

2015 ◽  
Vol 118 ◽  
pp. 81-94 ◽  
Author(s):  
Jean-Philippe Lambert ◽  
Monika Tucholska ◽  
Christopher Go ◽  
James D.R. Knight ◽  
Anne-Claude Gingras
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Interactome Mapping
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