[14] Detection of protein-protein interactions by protein fragment complementation strategies

doi:10.1016/S0076-6879(00)28399-7

Methods in Enzymology

Volume 328, 2000, Pages 208-230

https://doi.org/10.1016/S0076-6879(00)28399-7 Get rights and content

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This chapter presents the basic concept of protein fragment complementation assays (PCAs) and how they are designed, with particular attention to the system developed based on murine dihydrofolate reductase (mDHFR). It then discusses several applications of the assay, including a simple, large-scale library-versus-library screening strategy in Escherichia coli. The implementation of mammalian assays is discussed, including applications to the quantitative detection of induced protein interactions and allosteric transitions in intact cells. Finally, the generality of the PCA strategy is demonstrated with examples of assays that are designed on the basis of other enzymes including glycinamide ribonucleotide transformylase, aminoglycoside kinase, and hygromycin B kinase.

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Heterologous expression of active, native-folded protein in Escherichia coli is critical in both academic research and biotechnology settings. When expressing non-native recombinant proteins in E. coli, obtaining soluble and active protein can be challenging. Numerous techniques can be used to enhance a proteins solubility, and largely focus on either altering the expression strain, plasmid vector features, growth conditions, or the protein coding sequence itself. However, there is no one-size-fits-all approach for addressing issues with protein solubility, and it can be both time and labor intensive to find a solution. An alternative approach is to use the co-expression of chaperones to assist with increasing protein solubility. By designing a genetic system where protein solubility is linked to viability, the appropriate protein folding factor can be selected for any given protein of interest. To this end, we developed a Split Antibiotic Selection (SAS) whereby an insoluble protein is inserted in-frame within the coding sequence of the hygromycin B resistance protein, aminoglycoside 7″-phosphotransferase-Ia (APH(7″)), to generate a tripartite fusion. By creating this tripartite fusion with APH(7″), the solubility of the inserted protein can be assessed by measuring the level of hygromycin B resistance of the cells.
We demonstrate the functionality of this system using a known protein and co-chaperone pair, the human mitochondrial Hsp70 ATPase domain (ATPase⁷⁰) and its co-chaperone human escort protein (Hep). Insertion of the insoluble ATPase⁷⁰ within APH(7ʹʹ) renders the tripartite fusion insoluble and results in sensitivity to hygromycin B. Antibiotic resistance can be rescued by expression of the co-chaperone Hep which assists in the folding of the APH(7ʹʹ)-ATPase⁷⁰-APH(7ʹʹ) tripartite fusion and find that cellular hygromycin B resistance correlates with the total soluble fusion protein. Finally, using a diverse chaperone library, we find that SAS can be used in a pooled genetic selection to identify chaperones capable of improving client protein solubility.
The tripartite APH(7ʹʹ) fusion links the in vivo solubility of the inserted protein of interest to hygromycin B resistance. This construct can be used in conjunction with a chaperone library to select for chaperones that increase the solubility of the inserted protein. This selection system can be applied to a variety of client proteins and eliminates the need to individually test chaperone-protein pairs to identify those that increase solubility.
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Abnormal protein interactions are linked to many human diseases. To study protein-protein interactions, affinity purification, chemical cross-linking, protein fragment complementation assays, and two-hybrid screening are employed (Bruckner, Polge, Lentze, Auerbach, & Schlattner, 2009; Dunham, Mullin, & Gingras, 2012; Michnick, Remy, Campbell-Valois, Vallee-Belisle, & Pelletier, 2000; Tang & Bruce, 2009). In the past decade, proximity-dependent labeling methods have emerged as effective complementary approaches to classical affinity purification and mass spectrometry-based protein interaction mapping.
Protein-protein interactions are central to most cellular processes and their dysregulation has been related to the development of various diseases. Proximity-based labeling methods are used to identify the endogenous interaction partners of specific proteins of interest (POIs). The POI is fused to promiscuous enzymes, which generate reactive species in vivo and label proteins in close vicinity. APEX-based proximity labeling techniques utilize an engineered ascorbate peroxidase, which in the presence of H₂O₂ oxidizes biotin-phenol to short lived biotin-phenoxyl radicals that biotinylate nearby proteins. The biotinylated proteins are enriched by biotin affinity capture and identified by mass spectrometry. We devised an advanced method, RAPIDS, in which the peroxidase is physically separated from the POI and only a rapamycin-induced dimerization using the FRB-FKBP12 system brings the two proteins together. RAPIDS improves the specificity of APEX-based interactome analysis by strictly eliminating false positives. In this chapter, we describe this method in detail, with VAPB as a protein of interest and versions of APEX2 with different subcellular localizations. VAPB localizing to different cellular compartments, the endoplasmic reticulum and the inner nuclear membrane, yielded distinct sets of proximity partners as identified by RAPIDS.
Molecular Imaging of Protein-Protein Interactions and Protein Folding
2021, Molecular Imaging: Principles and Practice
In the last 2 decades, there has been significant interest in the field of reporter gene imaging, with the aim of determining the location(s), duration, and extent of gene expression within living subjects. An important application of this is in molecular imaging of interacting protein partners and protein conformational changes, an area that could pave the way to functional proteomics in living animals and provide a tool for whole-body evaluation of new pharmaceuticals targeted to modulate protein–protein interactions and protein misfolding. We review the three general methods currently available for imaging protein–protein interactions in living subjects using reporter genes, namely a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer system, and split reporter protein complementation and reconstitution strategies.
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Engineering precise control of enzymatic activity provides a powerful means to manipulate and understand biological processes. One approach to achieve a switch-like control over enzyme activity is to design a split enzyme, where the protein is separated into two polypeptides with each inactive fragment fused to inducible dimerization domains. The activity of the enzyme can be controlled by the addition of a small molecule, which causes the inducible dimerization domains to come together and reconstitute the split enzyme and its activity. In recent years, split enzymes have been designed for a variety of enzyme classes, and these synthetic molecular tools have enabled spatial and temporal dissection of biological processes in ways that were difficult previously. Here, we summarize key design principles and strategies to guide future split enzyme engineering efforts, using split enzymes generated from our research group as examples.
Dynamics of protein complex components
2019, Current Opinion in Chemical Biology
Identifying protein-protein interactions (PPIs) is necessary to understand the molecular mechanisms behind cellular processes. This task is complicated by the facts that many proteins can interact simultaneously (i.e. a protein complex) and may participate in more than one distinct complex. Because of this, a large number of combinatorial arrangements are possible, both of PPIs and complexes, making it a difficult task to identify all truly interacting proteins. Protein interactions also range from stable to highly transient assemblies, with lifetimes on the order of seconds [1]. Therefore, studies identifying PPIs must not only contend with the arrangement of proteins into PPIs and complexes, but the stability of the interactions as well. Because of the difficulty of the task, many approaches have been used to identify and study the dynamics of PPIs. In this review, we will summarize a number of the techniques currently used to identify protein-protein interactions, with a focus on recent developments.
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2019, Computational and Structural Biotechnology Journal
Citation Excerpt :
One of the primary goals of systems biology is to understand the functions of proteins from various organisms [1]. Some of the most widely used techniques to identify protein-protein interactions include yeast two-hybrid (Y2H) [2,3], protein-fragment complementation assay (PCA) [4], LUMIER [5], fluorescence resonance energy transfer (FRET) [6] etc. Mass spectrometry is a powerful tool for studying biomolecules such as proteins by their identification, quantification and further functional characterization [7–9].
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[14] Detection of protein-protein interactions by protein fragment complementation strategies

Publisher Summary

Cell

Cell

Cell

Trends Cell Biol.

Curr. Opin. Chem. Biol.

Curr. Opin. Biotechnol.

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J. Biol. Chem.

J. Biol. Chem.

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J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

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J. Biol. Chem.

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J. Biol. Chem.

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FEBS Lett.

Cell

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

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J. Mot. Biol.

J. Mol. Biol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Cell

Nature (London)

Nucleic Acids Res.

Science