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Stephen W. Michnick - One of the best experts on this subject based on the ideXlab platform.
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Combining the Optimized Yeast Cytosine Deaminase Protein Fragment Complementation Assay and an In Vitro Cdk1 Targeting Assay to Study the Regulation of the γ-Tubulin Complex.
Methods of Molecular Biology, 2020Co-Authors: Jacqueline Kowarzyk, Jackie Vogel, Michael J. Booth, Diala Abd-rabbo, Kristian Shulist, Conrad Hall, Stephen W. MichnickAbstract:: Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based Protein-Fragment Complementation Assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast γ-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase Assay using the γ-tubulin complex as a substrate.
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Synthesis of degenerated libraries of the ras-binding domain of raf and rapid selection of fast-folding and stable clones with the dihydrofolate reductase protein fragment Complementation Assay.
Methods of Molecular Biology, 2020Co-Authors: François-xavier Campbell-valois, Stephen W. MichnickAbstract:: The protein-engineering field is mainly concerned with the design of novel enzyme activities or folds and with understanding the fundamental sequence determinants of protein folding and stability. Much effort has been put into the design of methods to generate and screen libraries of polypeptides. Screening for the ability of proteins to bind with high affinity and/or specificity is most often approached with phage display technologies. In this chapter, we present an alternative to phage display, performed totally in vivo, based on the dihydrofolate reductase (DHFR) Protein-Fragment Complementation Assay (PCA). We describe the application of the DHFR PCA to the selection of degenerated sequences of the ras-binding domain (RBD) of raf for correct folding and binding to ras. Our screening system allows for enrichment of the libraries for the best-behaving sequences through iterative competition experiments, without the discrete library screening and expansion steps that are necessary in in vitro approaches. Moreover, the selected clones can be processed rapidly to purification by Ni-nitrilotriacetic acid (NTA) affinity chromatography in 96-well plates. Our methods are particularly suitable for the designing and screening of libraries aimed at studying sequence folding and binding determinants. Finally, it can be adapted for library-against-library screening, thus, allowing for coevolution of interacting proteins simultaneously.
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Dissecting the Contingent Interactions of Protein Complexes with the Optimized Yeast Cytosine Deaminase Protein-Fragment Complementation Assay.
CSH Protocols, 2016Co-Authors: Jacqueline Kowarzyk, Stephen W. MichnickAbstract:: Here, we present a detailed protocol for studying in yeast cells the contingent interaction between a substrate and its multisubunit enzyme complex by using a death selection technique known as the optimized yeast cytosine deaminase Protein-Fragment Complementation Assay (OyCD PCA). In yeast, the enzyme cytosine deaminase (encoded by FCY1) is involved in pyrimidine metabolism. The PCA is based on an engineered form of yeast cytosine deaminase optimized by directed evolution for maximum activity (OyCD), which acts as a reporter converting the pro-drug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a toxic compound that kills the cell. Cells that have OyCD PCA activity convert 5-FC to 5-FU and die. Using this Assay, it is possible to assess how regulatory subunits of an enzyme contribute to the overall interaction between the catalytic subunit and the potential substrates. Furthermore, OyCD PCA can be used to dissect different functions of mutant forms of a protein as a mutant can disrupt interaction with one partner, while retaining interaction with others. As it is scalable to a medium- or high-throughput format, OyCD PCA can be used to study hundreds to thousands of pairwise protein-protein interactions in different deletion strains. In addition, OyCD PCA vectors (pAG413GAL1-ccdB-OyCD-F[1] and pAG415GAL1-ccdB-OyCD-F[2]) have been designed to be compatible with the proprietary Gateway technology. It is therefore easy to generate fusion genes with the OyCD reporter fragments. As an example, we will focus on the yeast cyclin-dependent protein kinase 1 (Cdk1, encoded by CDC28), its regulatory cyclin subunits, and its substrates or binding partners.
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The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein-Protein Interactions.
CSH Protocols, 2016Co-Authors: Stephen W. Michnick, Christian R. Landry, Jacqueline Kowarzyk, Emmanuel D. Levy, Vincent MessierAbstract:: Protein-Fragment Complementation Assays (PCAs) can be used to study protein-protein interactions (PPIs) in any living cell, in vivo or in vitro, in any subcellular compartment or membranes. Here, we present a detailed protocol for performing and analyzing a high-throughput PCA screening to study PPIs in yeast, using dihydrofolate reductase (DHFR) as the reporter protein. The DHFR PCA is a simple survival-selection Assay in which Saccharomyces cerevisiae DHFR (scDHFR) is inhibited by methotrexate, thus preventing nucleotide synthesis and causing arrest of cell division. Complementation of cells with a methotrexate-insensitive murine DHFR restores nucleotide synthesis, allowing cell proliferation. The methotrexate-resistant DHFR has two mutations (L22F and F31S) and is 10,000 times less sensitive to methotrexate than wild-type scDHFR, but retains full catalytic activity. The DHFR PCA is sensitive enough for PPIs to be detected for open reading frame (ORF)-PCA fragments expressed off of their endogenous promoters.
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Dissection of Cdk1–cyclin complexes in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Michael J. Booth, Jackie Vogel, Diala Abd-rabbo, Conrad Hall, Jacqueline Kowarzyk Moreno, Daici Chen, Stephen W. MichnickAbstract:Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin–Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin–Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a Protein-Fragment Complementation Assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3–Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes.
J Reiser - One of the best experts on this subject based on the ideXlab platform.
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Rapid titration of retroviral vectors using a β-lactamase protein fragment Complementation Assay
Gene Therapy, 2013Co-Authors: Wu Ou, M P Marino, C Lu, J ReiserAbstract:The availability of rapid and quantitative titration Assays for retroviral vectors is important, especially in the context of clinical applications. In this report, we describe a novel Assay to titrate lentiviral and gamma retroviral vectors. This rapid Assay is based on protein fragment Complementation involving the N-terminal (Bla1) and the C-terminal (Bla2) fragments of TEM-1 β-lactamase (BLAK). The Bla1 protein fragment is incorporated in the vector's envelope during vector production. Bla1-bearing vectors are titrated on Bla2-expressing cells. Upon transduction, Bla1 and Bla2 heterodimerize and restore BLAK's enzymatic function. The enzymatic activity of BLAK is quantified by flow cytometry using the green fluorescent CCF2/AM substrate, which is converted into a blue fluorescent product. The enzymatic conversion of the CCF2/AM substrate was found to be directly related to vector entry, as a neutralizing antibody completely blocked the conversion. The titers obtained using this rapid Assay correlated well with the titers measured by functional transduction Assays. The whole Assay can be finished within 8 h. Thus, it is considerably less time consuming compared with other transduction-based titration Assays for lentiviral and gamma retroviral vectors.
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Rapid titration of retroviral vectors using a β-lactamase protein fragment Complementation Assay
Gene Therapy, 2012Co-Authors: Wu Ou, M P Marino, C Lu, J ReiserAbstract:Rapid titration of retroviral vectors using a β-lactamase protein fragment Complementation Assay
Eiry Kobatake - One of the best experts on this subject based on the ideXlab platform.
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Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein–Protein Complexes in Living Cells
Analytical Chemistry, 2016Co-Authors: Tatsuhiko Naoki, Eiry KobatakeAbstract:The split SNAP-tag Protein-Fragment Complementation Assay (PCA) is a useful tool for imaging protein–protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein–protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein–protein complex. To track both proteins forming a protein–protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein–protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein–protein complex and subsequent tracking of eac...
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development of a split snap clip double labeling system for tracking proteins following dissociation from protein protein complexes in living cells
Analytical Chemistry, 2016Co-Authors: Tatsuhiko Naoki, Eiry KobatakeAbstract:The split SNAP-tag Protein-Fragment Complementation Assay (PCA) is a useful tool for imaging protein–protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein–protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein–protein complex. To track both proteins forming a protein–protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein–protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein–protein complex and subsequent tracking of eac...
Per-Åke Nygren - One of the best experts on this subject based on the ideXlab platform.
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Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment Complementation Assay.
New Biotechnology, 2009Co-Authors: P-a Löfdahl, Olof Nord, Lars Janzon, Per-Åke NygrenAbstract:Protein fragment Complementation Assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The pr ...
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Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment Complementation Assay.
New biotechnology, 2009Co-Authors: P-a Löfdahl, Olof Nord, Lars Janzon, Per-Åke NygrenAbstract:Protein fragment Complementation Assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naïve 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the beta-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition Assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PCA is a promising methodology for stringent selection of binders from complex naïve libraries to yield high affinity reagents with selective target binding characteristics.
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Fluorescent detection of β-lactamase activity in living Escherichia coli cells via esterase supplementation
Fems Microbiology Letters, 2004Co-Authors: Olof Nord, Anna Gustrin, Per-Åke NygrenAbstract:The TEM-1 β-lactamase protein fragment Complementation Assay was investigated for its applicability in affinity protein-based interaction studies in Escherichia coli, using an affibody-based model system. Results from co-transformation experiments showed that an ampicillin resistant phenotype was specifically associated with cognate affibody–target pairings. Attempts to monitor β-lactamase Complementation in vitro with the fluorescent β-lactamase substrates CCF2/AM and CCF2 showed that E. coli lacks an esterase activity necessary for activation of the esterified and membrane-permeable CCF2/AM form of the substrate. Interestingly, supplementation of the Assay reaction with a purified fungal lipase (cutinase) resulted in efficient activation of CCF2/AM in vitro. Further, periplasmic expression of cutinase allowed for fluorescent discrimination between β-lactamase positive and negative living E. coli cells using the CCF2/AM substrate, which should open the way for novel applications for this prokaryotic host in protein interaction studies.
Wu Ou - One of the best experts on this subject based on the ideXlab platform.
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Rapid titration of retroviral vectors using a β-lactamase protein fragment Complementation Assay
Gene Therapy, 2013Co-Authors: Wu Ou, M P Marino, C Lu, J ReiserAbstract:The availability of rapid and quantitative titration Assays for retroviral vectors is important, especially in the context of clinical applications. In this report, we describe a novel Assay to titrate lentiviral and gamma retroviral vectors. This rapid Assay is based on protein fragment Complementation involving the N-terminal (Bla1) and the C-terminal (Bla2) fragments of TEM-1 β-lactamase (BLAK). The Bla1 protein fragment is incorporated in the vector's envelope during vector production. Bla1-bearing vectors are titrated on Bla2-expressing cells. Upon transduction, Bla1 and Bla2 heterodimerize and restore BLAK's enzymatic function. The enzymatic activity of BLAK is quantified by flow cytometry using the green fluorescent CCF2/AM substrate, which is converted into a blue fluorescent product. The enzymatic conversion of the CCF2/AM substrate was found to be directly related to vector entry, as a neutralizing antibody completely blocked the conversion. The titers obtained using this rapid Assay correlated well with the titers measured by functional transduction Assays. The whole Assay can be finished within 8 h. Thus, it is considerably less time consuming compared with other transduction-based titration Assays for lentiviral and gamma retroviral vectors.
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Rapid titration of retroviral vectors using a β-lactamase protein fragment Complementation Assay
Gene Therapy, 2012Co-Authors: Wu Ou, M P Marino, C Lu, J ReiserAbstract:Rapid titration of retroviral vectors using a β-lactamase protein fragment Complementation Assay
