We would like to kindly acknowledge Prof. Dr. Lothar Willmitzer for his involvement in the project, productive discussions, and great supervision. We are grateful to Dr. Daniel Veyel for helping with proteomic MS measurements. We appreciate Mrs. &#196;nne Michaelis who provided us invaluable technical help with LC-MS measurements. Furthermore, we would like to thank Dr. Monika Kosmacz and Dr. Ewelina Soko&#322;owska for their help and involvement in the work on the original manuscript, and to Weronika Jasi&#324;ska for technical support.

<ol>
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The authors have nothing to disclose.

The presented protocol allows parallel identification of PP and PM complexes of a target protein. From cloning to final results, the experiment can be completed in as little as 8-12 weeks. Complete AP takes about 4-6 h for a set of 12 to 24 samples, rendering our protocol suitable for mid-throughput analysis.
The protocol, despite being overall straightforward, has a number of critical steps. (i) Sufficient amount of input protein and affinity beads is crucial to reach a dynamic range of metabolite detection. Efficient cell lysis is therefore a crucial step in the procedure. Poor protein yields can be a consequence of insufficient pulverization of the material or of suboptimal lysis-buffer/material ratio. (ii) Care should be taken that used reagents are MS-friendly. Strong detergents, glycerol, or excessive amounts of salt should be avoided as they interfere with MS detection. (iii) Agarose beads should not be over-dried during washing steps, and when using a vacuum manifold it is important to apply a slow flow rate so as not to destroy the beads or affect complex stability.
There are some important possible modifications to the presented protocol: (i) We use the constitutive CaMV35S promoter to maximize the amount of bait protein. Overexpression, while very useful, can have serious effects on cell homeostasis28 and lead to the formation of physiologically irrelevant interactions. Expression of tagged proteins using native promoters and where possible in a loss-of-function background is considered superior for retrieving true biological interactors. For proteins normally not expressed in plant cell cultures, a plant background may prove necessary to identify relevant interactors. (ii) When working with membrane proteins, the lysis buffer needs to be supplemented with an MS-compatible detergent. (iii) Introduction of a second affinity-purification step could improve false-positives to true-positives ratio and eliminate the need for EV controls29. A novel tandem tag with two independent protease-cleavage sites presents an attractive alternative to the size-exclusion chromatography step added by Maeda et al. 201411, which is both laborious and time consuming.
The most serious drawback of the AP is the high rate of false positives. The reasons are numerous. Constitutive overexpression was already mentioned. Another source of physiologically irrelevant interactions, unless working with isolated organelles, is preparation of whole-cell lysates containing mixtures of proteins and metabolites from different subcellular compartments. Subcellular localization should be used to filter for true interactors. Nevertheless, the majority of false positives result from unspecific binding between proteins and agarose resins. Introduction of a second purification step, as described above, offers the best solution to the problem, however comes at the cost of time and throughput. Moreover, weaker interaction may be lost as the protocol lengthens. Another caveat of AP is that despite the comprehensive information it provides about the interactome of a target protein, differentiating between direct and indirect targets of the baited protein is impossible. Targeted bimolecular approaches are needed to confirm interactions.
AP coupled with MS-based metabolomics was used to study protein- complexes in S. cerevisiae12. This work, together with our earlier observation13 that, similarly to lipids, polar and semi-polar compounds remain bound to protein complexes isolated from cellular lysates, provided conceptual groundwork for the presented protocol. Our protocol is characterized by three unique points: (i) In contrast to the yeast work12, it demonstrates that AP is suitable for retrieving not only hydrophobic but also hydrophilic protein ligands. (ii) By introducing a three-in-one extraction protocol, a single AP can be used to study protein and metabolite interactors of the bait protein. (iii) We adapted the protocol to plant cells.
Future efforts will focus on creating a novel tandem tag with two independent protease-cleavage sites. We would also like to explore suitability of the protocol to low-abundance small molecules such as plant hormones.

The method described here aims at the identification of metabolite and protein partners of a protein of choice in near-in-vivo cellular lysate conditions. It has been speculated that many more metabolites than characterized today have an important regulatory function1. Metabolites can act as biological switches, changing the activity, functionality, and/or localization of their receptor proteins2,3,4. In the last decade several breakthrough methods, enabling identification of PMI in vivo or in near-in-vivo conditions, have been developed5. Available approaches can be separated into two groups. The first group comprises techniques that start with a known-metabolite bait in order to trap novel protein partners. Methods include affinity chromatography6, drug affinity responsive target-stability assay7, chemo-proteomics8, and thermal proteome profiling9. The second group consists of a single method that starts with a known protein in order to identify small-molecule ligands10,11.
AP coupled with MS-based lipidomics was used to analyze protein-lipid complexes in Saccharomyces cerevisiae12. As a starting point, the authors used yeast strains expressing 21 enzymes involved in ergosterol biosynthesis and 103 kinases fused to a tandem-affinity purification (TAP) tag. 70% of the enzymes and 20% of the kinases were found to bind different hydrophobic ligands, shedding light into the intricate protein-lipid interaction network.
Previously, we could demonstrate that, similarly to lipids, polar and semi-polar compounds also remain bound to protein complexes isolated from the cellular lysates13. Based on these findings, we decided to optimize the AP method published previously10,11 for plant cells and hydrophilic compounds14. For this purpose, we used TAP vectors described by Van Leene et al. 2010, successfully used in plant PPI studies15. To shorten the time required to obtain transgenic lines, we decided on Arabidopsis cell cultures. We employed a one-step methyl tert-butyl ether, (MTBE)/methanol/water extraction method, allowing the characterization of proteins (pellet), lipids (organic phase), and hydrophilic metabolites (aqueous phase)16 in a single affinity-purification experiment. EV control lines were introduced to exclude false positives, e.g. proteins binding to the tag alone. As proof of concept we tagged three (of the five) nucleoside diphosphate kinases present in the Arabidopsis genome (NDPK1-NDPK3). Among other findings, we could demonstrate that NDPK1 interacts with glutathione S-transferase and glutathione. Consequently we could prove that NDPK1 is subjected to glutathionylation14.
To sum up, the presented protocol is an important tool for characterizing protein-protein and protein-small-molecule interaction networks and constitutes a major advance over existing methods.

<table>
	<tbody>
		<tr>
			<td>Murashige and Skoog Basal Salts with minimal organics</td>
			<td>Sigma-Aldrich</td>
			<td>M6899</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Naphthylacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N1641</td>
			<td></td>
		</tr>
		<tr>
			<td>Kinetin solution</td>
			<td>Sigma-Aldrich</td>
			<td>K3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>2189.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>3609</td>
			<td></td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Sigma-Aldrich</td>
			<td>S6776</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>E-64 protease inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>E3132</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P9599</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Sigma-Aldrich</td>
			<td>S6508</td>
			<td></td>
		</tr>
		<tr>
			<td>AcTEV Protease</td>
			<td>Thermo Fischer Scientific</td>
			<td>12575015</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotiphorese&#160;Gel 30 (37,5:1)</td>
			<td>Carl Roth</td>
			<td>3029.2</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Fischer Scientific</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>SBP Tag Antibody (SB19-C4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-101595</td>
			<td></td>
		</tr>
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			<td>Goat anti-mouse IgG-HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>B6916</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C Mix, Mass Spec Grade</td>
			<td>Promega&#160;</td>
			<td>V5071</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma-Aldrich</td>
			<td>T8656</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr>
			<td>MTBE&#160;</td>
			<td>Biosolve</td>
			<td>138906</td>
			<td></td>
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		<tr>
			<td>Methanol&#160;</td>
			<td>Biosolve</td>
			<td>136806</td>
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		<tr>
			<td>Water</td>
			<td>Biosolve</td>
			<td>232106</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Biosolve</td>
			<td>12006</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid&#160;</td>
			<td>Biosolve</td>
			<td>202341</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid&#160;</td>
			<td>Biosolve</td>
			<td>69141</td>
			<td></td>
		</tr>
		<tr>
			<td>Unimax 2010 Platform Shaker</td>
			<td>Heidolph</td>
			<td>5421002000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh (Wire diameter 34 &#181;M, thickness 55 &#181;M, open area 14%)</td>
			<td>Prosepa</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Funnel, 47 mm, 300 ml</td>
			<td>Restek</td>
			<td>KT953751-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Bottle Top 500 mL 0,2 &#181;M Pes St</td>
			<td>VWR International GmbH</td>
			<td>514-0340</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Mill MM 400</td>
			<td>Retsch GmbH</td>
			<td>207450001</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Sepharose 6 Fast Flow</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-0969-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobicol &#34;&#34;Classic&#34;&#34; with 2 different screw caps without filters</td>
			<td>MoBiTec GmbH</td>
			<td>M1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (small) 35 &#181;M pore size, for Mobicol M 1002, M1003, M1050 &#38; M1053</td>
			<td>MoBiTec GmbH</td>
			<td>M513515</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable Speed Tube Rotator SB 3</td>
			<td>Carl Roth</td>
			<td>Y550.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary dishes for rotators SB 3</td>
			<td>Carl Roth</td>
			<td>Y555.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Resprep 24-Port SPE Manifolds</td>
			<td>Restek&#160;</td>
			<td>26080</td>
			<td></td>
		</tr>
		<tr>
			<td>Finisterre C18/17% SPE Columns 100mg / 1ml</td>
			<td>Teknokroma</td>
			<td>TR-F034000</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler&#160;Vials</td>
			<td>Klaus Trott Chromatographie-Zubeh&#246;r</td>
			<td>40 11 01 740</td>
			<td></td>
		</tr>
		<tr>
			<td>Acclaim PepMap 100 C18 LC Column</td>
			<td>Thermo Fischer Scientific</td>
			<td>164534</td>
			<td></td>
		</tr>
		<tr>
			<td>EASY-nLC 1000 Liquid Chromatograph</td>
			<td>Thermo Fischer Scientific</td>
			<td>LC120</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>IQLAAEGAAPFALGMBDK</td>
			<td></td>
		</tr>
		<tr>
			<td>Acquity UPLC system&#160;</td>
			<td>Waters&#160;</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC HSS C18 Column, 100A, 1.8 &#181;M, 2.1 mM X 100 mM, 1/pkg</td>
			<td>Waters&#160;</td>
			<td>186003533</td>
			<td></td>
		</tr>
		<tr>
			<td>High-power ultrasonic cleaning baths for aqueous cleaning solutions</td>
			<td>Bandelin</td>
			<td>RK 31</td>
			<td></td>
		</tr>
		<tr>
			<td>Genedata Expressionist</td>
			<td>Genedata</td>
			<td>NaN</td>
			<td></td>
		</tr>
		<tr>
			<td>Xcalibur Software</td>
			<td>Thermo Fischer Scientific</td>
			<td>NaN</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>NaN</td>
			<td>NaN</td>
			<td></td>
		</tr>
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2 in 1: one-step affinity purification for the parallel analysis of protein-protein and protein-metabolite complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

In the original study, three A. thaliana NDPK genes were overexpressed in PSB-L cell suspension cultures under the control of the constitutive 35S promoter14 (Figure 1). Tandem affinity tag was fused to either carboxy- or amino-terminal end of a bait protein. The affinity-purified complexes were subjected to MTBE/methanol/water extraction16. Affinity-pulled proteins and small molecules were identified using MS (Tables S2 and S3).
To correct for false positives, blank samples were used to exclude small-molecule contaminants from the chemicals and laboratory consumables. Furthermore, metabolites and proteins that bind to either an affinity tag or resin alone were accounted for by using EV control lines.To retrieve true positives, two-tailed non-paired Student&#39;s t-test and Benjamini &#38; Hochberg false discovery rate correction was applied to identify metabolites (Table S4) and proteins (Table S5) significantly enriched in the NDPKs AP experiments (N- and C-terminally tagged NDPKs) in comparison to the EV control lines (FDR &#60; 0.1). Note that in the previous work, we used absence/presence criteria to delineate protein and small-molecule interactors.
Representative results are given for NDPK1, while metabolite data focus on dipeptides, a novel class of the small-molecule regulators studied in our group. Proteomic analysis revealed 26 putative protein partners of NDPK1. By further filtering for proteins co-localized in the same subcellular compartment as NDPK1 (cytosol), the list narrowed down to 13 putative protein interactors. Among the identified proteins were glutathione S-transferase, two elongation initiation factors, tubulin, and aconitate hydratase. Metabolomic analysis revealed four dipeptides Val-Leu, Ile-Glu, Leu-Ile, and Ile-Phe that specifically co-eluted with NDPK1 (Figure 2). Note that all four dipeptides share a hydrophobic residue in their N-terminus, suggesting shared binding specificity.
To look for known protein-protein and protein-metabolite complexes we queried 13 identified proteins and four dipeptides against the Stitch database25 (Figure 3). Several observations could be made: (i) None of the interactors was previously reported for NDPK1. (ii) APX1 ortholog was reported to interact with aldehyde dehydrogenase family member ALDH7B4, while translation initiation factor FBR12 with another translation initiation factor encoded by gene AT2G40290. (iii) The identified dipeptides have no reported protein partners. Co-eluted dipeptides were not reported earlier as associated to any retrieved plant protein. However, they play important roles in other organisms: Leu-Ile, e.g., has a neurotrophin-activating effect in a human cell line26. Note that the experiment does not allow identifying the exact topology of the system. For example, a dipeptide may interact directly with NDPK1 but may well be related to any of the co-purified proteins.
Taken together, our results show that the established procedure, employing AP together with mass spectrometry, facilitates identification of protein-protein and protein-small-molecule interactors and helps generate extensive information about the interactome of the target protein.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57720/57720fig1.jpg" /> 
Figure 1. Scheme of AP-MS workflow. (A) Preparation of a native soluble fraction from plant cell culture. (B) Next steps in the AP procedure. After loading the sample onto the column, the protein of interest (POI) fused to a TAP tag binds to the IgG antibody immobilized on the agarose beads. Washing of the column facilitates removal of unbound proteins and metabolites. After performing AcTEV cleavage, POI protein-metabolite complexes are eluted. (C) Separation of complexes into protein and metabolite fraction followed by semi-quantitative MS analysis. Part of this Figure is reproduced from Luzarowski et al. 201714. <a href="/files/ftp_upload/57720/57720fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57720/57720fig2.jpg" /> 
Figure 2. Dipeptides specifically co-eluting with NDPK1. Average intensities of four dipeptides Val-Leu (A), Ile-Glu (B), Leu-Ile (C), and Ile-Phe (D) measured in AP experiment were plotted. All four dipeptides show significant enrichment in NDPK1 samples compared to EV control (asterisks represent FDR &#60; 0.1). Error bars represent standard error for 6 measurements (3 replicates of N- and 3 of C-terminally tagged proteins). <a href="/files/ftp_upload/57720/57720fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57720/57720fig3.jpg" /> 
Figure 3. Interaction network of all molecules co-eluting with NDPK1, queried against STITCH database considering only previous experimental and database evidences (confidence &#62; 0.2). Higher confidence indicates higher chances of interaction and is calculated based on the deposited data. <a href="/files/ftp_upload/57720/57720fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table S1. MaxQuant output table &#34;parameters.txt&#34;. Table includes threshold values for identification and quantification, as well as information about the databases used. <a href="/files/ftp_upload/57720/Table_S1.xlsx">Please click here to download this file.</a>
Table S2. Information from MaxQuant output table &#34;proteinGroups.txt&#34;. Table contains a list of all identified protein groups, intensities, and additional information such as number of unique peptides and score. <a href="/files/ftp_upload/57720/Table_S2.xlsx">Please click here to download this file.</a>
Table S3. Output file containing analysis of polar metabolites. Table contains a list of all identified mass features characterized by specific m/z, RT and intensity. <a href="/files/ftp_upload/57720/Table_S3.xlsx">Please click here to download this file.</a>
Table S4. Dipeptides found in AP samples in which NDPK1, NDPK2 or NDPK3 were used as bait. Dipeptides present in blank samples were excluded from the list. Two independent lines (tagged in either N- or C-terminus) for each NDPK were run in triplicate. Student&#39;s t-test and further correction of p-value using Benjamini &#38; Hochberg method were used to determine significantly enriched interactor partners of NDPKs (FDR &#60; 0.1). Given is &#916;RT calculated in relation to the reference compounds and &#916;ppm in relation to the monoisotopic mass given in Metlin27. <a href="/files/ftp_upload/57720/Table_S4.xlsx">Please click here to download this file.</a>
Table S5. Proteins co-purified with NDPK1. Two independent lines (tagged in either N- or C-terminus) for each NDPK were run in triplicate. Student&#39;s t-test and further correction of p-value using Benjamini &#38; Hochberg method were used to determine significantly enriched interactor partners of NDPKs (FDR &#60; 0.1). <a href="/files/ftp_upload/57720/Table_S5.xlsx">Please click here to download this file.</a>

Watch this Scientific Journal Video about 2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes at JoVE.com

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

2-1-one-step-affinity-purification-for-parallel-analysis-protein

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.2K Views.  Max Planck Institute of Molecular Plant Physiology. Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Video: 2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...