scholarly journals A novel recombinant DNA system for high efficiency affinity purification of proteins in Saccharomyces cerevisiae

2015 ◽  
Author(s):  
Brian H Carrick ◽  
Lixuan HaO ◽  
Philip J Smaldino ◽  
David R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Efficiency ◽  
Recombinant Dna ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Single Step ◽  
Open Reading Frames ◽  
Tev Protease ◽  
Number Of Factors ◽  
Endogenous Proteins

Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. The tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background in a single step. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent will allow isolation of rare or unstable protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or chromatin-associated assemblies.

scholarly journals A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

2015 ◽  
Vol 6 (3) ◽  
pp. 573-578 ◽  
Author(s):  
Brian H. Carrick ◽  
Linxuan Hao ◽  
Philip J. Smaldino ◽  
David R. Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Efficiency ◽  
Recombinant Dna ◽  
Affinity Purification

scholarly journals Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 103-118 ◽  
Author(s):  
Janet R Mullen ◽  
Vivek Kaliraman ◽  
Samer S Ibrahim ◽  
Steven J Brill
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Stability ◽  
Protein Complexes ◽  
Ring Finger ◽  
Dna Helicase ◽  
Open Reading Frames ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Helicases ◽  
Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits

2004 ◽  
Vol 32 (6) ◽  
pp. 899-903 ◽  
Author(s):  
K.K. Lee ◽  
P. Prochasson ◽  
L. Florens ◽  
S.K. Swanson ◽  
M.P. Washburn ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Identification ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Large Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Chromatin Remodelling Complex ◽  
Novel Proteins ◽  
Multidimensional Protein Identification Technology ◽  
Covalent Histone Modifications

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic changes are the ATP-dependent Swi/Snf chromatin-remodelling complex and the SAGA histone acetyltransferase complex. To understand fully the mechanism by which these large protein complexes perform their functions in the cell, it is crucial that all the subunits of these complexes are identified. In an attempt to identify new subunits associated with SAGA and Swi/Snf, we used tandem affinity purification, followed by a multidimensional protein identification technology to analyse the subunit composition. Our analysis identified two novel proteins, one associated with SAGA, YPL047W (Sgf11), and another associated with Swi/Snf, Rtt102.

scholarly journals Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria

2006 ◽  
Vol 72 (7) ◽  
pp. 5027-5036 ◽  
Author(s):  
Robert M. Q. Shanks ◽  
Nicky C. Caiazza ◽  
Shannon M. Hinsa ◽  
Christine M. Toutain ◽  
George A. O'Toole
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Efficiency ◽  
Opportunistic Pathogen ◽  
Single Step ◽  
Expression Vectors ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Recombination Proteins ◽  
Wide Range

ABSTRACT A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.

Protein–protein interactions

2010 ◽  
Vol 38 (4) ◽  
pp. 875-878 ◽  
Author(s):  
Mike P. Williamson ◽  
Michael J. Sutcliffe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Present Article ◽  
High Throughput ◽  
Protein Interactions ◽  
Metabolic Pathway ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Good Evidence ◽  
Protein Protein Interactions ◽  
Two Hybrid

In the present article, we describe the two standard high-throughput methods for identification of protein complexes: two-hybrid screens and TAP (tandem affinity purification) tagging. These methods have been used to characterize the interactome of Saccharomyces cerevisiae, showing that the majority of proteins are part of complexes, and that complexes typically consist of a core to which are bound ‘party’ and ‘dater’ proteins. Complexes typically are merely the sum of their parts. A particularly interesting type of complex is the metabolon, containing enzymes within the same metabolic pathway. There is reasonably good evidence that metabolons exist, but they have not been detected using high-thoughput assays, possibly because of their fragility.

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.

A pipeline for systematic yeast 2-hybrid matricial screening in liquid culture.

2019 ◽  
Author(s):  
Monachello Dario ◽  
Guillaumot Damien ◽  
Lurin Claire
Keyword(s):  
High Throughput ◽  
Protein Interaction ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Operating Cost ◽  
Open Reading Frames ◽  
Critical Element ◽  
Interactome Mapping ◽  
Interaction Map ◽  
Split Ubiquitin

Abstract Physical interactions mediated by proteins are a critical element of biological systems, and the analysis of interaction partners can provide valuable hints about unknown functions of a protein. Two major classes of experimental approaches are used for protein interaction mapping: analysis of direct interactions using binary methods such as yeast two-hybrid (Y2H) or split ubiquitin, and analysis of protein complexes through affinity purification followed by mass spectrometry. Thanks to his flexibility to low- and high-throughput approaches and a low operating cost the Y2H assay is widely used for high-throughput interaction mapping experiments. Moreover, it has now been shown that high-throughput methods can produce highly reliable interactome datasets1 2 3 4. Notably, in 2011 a proteome-wide binary protein-protein interaction map of the plant Arabidopsis thaliana 5 (Arabidopsis Interactome Mapping project – AIM) was described using a high-throughput binary interactome mapping pipeline based on the Y2H system and using a collection of ~8,000 open reading frames (8k_space). Here we describe a liquid pipeline for a high-throughput binary protein–protein Y2H screen of a pool of 50 proteins used as baits against a collection of ~12,000 Arabidopsis proteins encoded by sequence-verified ORFs (12k_space)6 7.

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