Name: rapid one-step enzymatic synthesis and all-aqueous purification of trehalose analogues
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This work was funded by a grant from the National Institutes of Health (R15 AI117670) to B.M.S and P.J.W, as well as a Cottrell College Scholar Award from the Research Corporation (20185) to P.J.W. L.M.M. was supported by a Provost's Fellowship from CMU.

1. Expression and Purification of TreT from Top10 E. coli
NOTE: Please contact the authors to request the TreT-expressing E. coli strain (pBAD TreT plasmid, containing the T. tenax tret gene under the control of the AraC protein, transformed into Top10 E. coli 19) and the accompanying material transfer agreement. The following protocol typically gives a protein yield of approximately 4 mg/L.
<ol>
	<li>Prepare a 3 mL overnight culture of TreT-expressing ...

<ol>
	<li>Elbein, A. D., Pan, Y. T., Pastuszak, I., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+on+trehalose:+a+multifunctional+molecule.">New insights on trehalose: a multifunctional molecule.</a> Glycobiology. 13, 17-27 (2003).</li><li>Tournu, H., Fiori, A., Van Dijck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relevance+of+trehalose+in+pathogenicity:+some+general+rules,+yet+many+exceptions.">Relevance of trehalose in pathogenicity: some general rules, yet many exceptions.</a> PLoS Pathog. 9, 1003447 (2013).</li><li>Ohtake, S., Wang, Y. 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Ecol. 18, 107-110 (1990).</li><li>Kubota, M., Ohnishi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Glycoenzymes. , (2000).</li><li>Walmagh, M., Zhao, R., Desmet, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+analogues:+latest+insights+in+properties+and+biocatalytic+production.">Trehalose analogues: latest insights in properties and biocatalytic production.</a> Int. J. Mol. Sci. 16, 13729-13745 (2015).</li><li>Kim, H. -. M., Chang, Y. -. K., Ryu, S. -. I., Moon, S. -. G., Lee, S. -. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+mycobacterial+trehalome+with+bioorthogonal+chemistry.">Probing the mycobacterial trehalome with bioorthogonal chemistry.</a> J. Am. Chem. Soc. 134, 16123-16126 (2012).</li><li>Rose, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+biological+evaluation+of+trehalose+analogs+as+potential+inhibitors+of+mycobacterial+cell+wall+biosynthesis.">Synthesis and biological evaluation of trehalose analogs as potential inhibitors of mycobacterial cell wall biosynthesis.</a> Carbohydr. Res. 337, 105-120 (2002).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+trehalose-based+compounds+and+their+inhibitory+activities+against+Mycobacterium+smegmatis.">Synthesis of trehalose-based compounds and their inhibitory activities against Mycobacterium smegmatis.</a> Bioorg. Med. Chem. 12, 6397-6413 (2004).</li><li>Gobec, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+synthesis,+biochemical+evaluation+and+antimycobacterial+action+of+phosphonate+inhibitors+of+antigen+85C,+a+crucial+enzyme+involved+in+biosynthesis+of+the+mycobacterial+cell+wall.">Design, synthesis, biochemical evaluation and antimycobacterial action of phosphonate inhibitors of antigen 85C, a crucial enzyme involved in biosynthesis of the mycobacterial cell wall.</a> Eur. J. Med. Chem. 42, 54-63 (2007).</li><li>Sarpe, V. A., Kulkarni, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regioselective+protection+and+functionalization+of+trehalose.">Regioselective protection and functionalization of trehalose.</a> Trends in Carbohydr. Res. 5, 8-33 (2013).</li><li>Chaube, M. A., Kulkarni, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereoselective+construction+of+1,1-alpha,alpha-glycosidic+bonds.">Stereoselective construction of 1,1-alpha,alpha-glycosidic bonds.</a> Trends in Carbohydr. Res. 4, 1-19 (2013).</li><li>Leigh, C. D., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+studies+toward+Mycobacterium+tuberculosis+sulfolipid-I.">Synthetic studies toward Mycobacterium tuberculosis sulfolipid-I.</a> J. Org. Chem. 73, 1008-1017 (2008).</li><li>Chaen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+enzymatic+synthesis+of+disaccharide,+alpha-D-galactosyl-alpha-D-glucoside,+by+trehalose+phosphorylase+from+Thermoanaerobacter+brockii.">Efficient enzymatic synthesis of disaccharide, alpha-D-galactosyl-alpha-D-glucoside, by trehalose phosphorylase from Thermoanaerobacter brockii.</a> J. Appl. Glycosci. 48, 135-137 (2001).</li><li>Vander Borght, J., Soetaert, W., Desmet, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+acceptor+specificity+of+trehalose+phosphorylase+for+the+production+of+trehalose+analogs.">Engineering the acceptor specificity of trehalose phosphorylase for the production of trehalose analogs.</a> Biotechnol. Progr. 28, 1257-1262 (2012).</li><li>Kouril, T., Zaparty, M., Marrero, J., Brinkmann, H., Siebers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+trehalose+synthesizing+pathway+in+the+hyperthermophilic+Crenarchaeon+Thermoproteus+tenax:+the+unidirectional+TreT+pathway.">A novel trehalose synthesizing pathway in the hyperthermophilic Crenarchaeon Thermoproteus tenax: the unidirectional TreT pathway.</a> Arch. Microbiol. 190, 355-369 (2008).</li><li>Urbanek, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoenzymatic+synthesis+of+trehalose+analogues:+rapid+access+to+chemical+probes+for+investigating+mycobacteria.">Chemoenzymatic synthesis of trehalose analogues: rapid access to chemical probes for investigating mycobacteria.</a> ChemBioChem. 15, 2066-2070 (2014).</li><li>Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+Huisgen+cycloaddition+process:+copper(I)-catalyzed+regioselective+"ligation"+of+azides+and+terminal+alkynes.">A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes.</a> Angew. Chem. Int. Ed. 41, 2596-2599 (2002).</li><li>Torn&#248;e, C. W., Christensen, C., Meldal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidotriazoles+on+solid+phase:+[1,2,3]-triazoles+by+regiospecific+copper(I)-catalyzed+1,3-dipolar+cycloadditions+of+terminal+alkynes+to+azides.">Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.</a> J. Org. Chem. 67, 3057-3064 (2002).</li><li>Kalscheuer, R., Weinrick, B., Veeraraghavan, U., Besra, G. S., Jacobs, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose-recycling+ABC+transporter+LpqY-SugA-SugB-SugC+is+essential+for+virulence+of+Mycobacterium+tuberculosis.">Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 21761-21766 (2010).</li></ol>

Trehalose analogues have the potential to impact various fields, from preservation of food and pharmaceuticals to diagnosis and treatment of microbial infections 6. Existing multistep chemical synthesis methods are useful for producing complex trehalose analogues with multiple sites of modification (e.g., naturally occurring complex mycobacterial glycolipids). However, these methods are invariably lengthy and inefficient, even when applied to the synthesis of comparatively simple monosubs...

Trehalose is a symmetrical non-reducing disaccharide consisting of two glucose moieties that are joined by a 1,1-&#945;,&#945;-glycosidic bond (Figure 1A). While trehalose is absent from humans and other mammals, it is found commonly in bacteria, fungi, plants, and invertebrates 1. The primary role of trehalose in most organisms is to protect against environmental stresses, such as desiccation 1. In addition, some human pathogens require trehalose for virulence, including the tuberculosis-causing Mycobacterium tuberculosis, which utilizes trehalose as a mediator of cell envelope biosynthesis and as a building block for the construction of immunomodulatory glycolipids 2.
<img alt="Figure 1" src="/files/ftp_upload/54485/54485fig1.jpg" /> 
Figure 1: Trehalose and trehalose analogues. (A) Structures of natural trehalose and an unnatural trehalose analogue, where X is a structural modification. (B) Examples of trehalose analogues reported in the literature that have potential applications in biopreservation and bioimaging.
Due to its unique structure and physiological functions, trehalose has drawn significant attention for use in bio(techno)logical and biomedical applications 3. The protective properties of trehalose observed in nature&#8212;e.g., its striking ability to help sustain life in &#34;resurrection&#34; plants that have undergone extreme dehydration 4&#8212;have spurred its extensive use in biopreservation applications. Trehalose has been used to preserve a wide array of biological samples, such as nucleic acids, proteins, cells, and tissues 3. For instance, trehalose is used as a stabilizing additive in a number of pharmaceuticals that are on the market, including several anti-cancer monoclonal antibodies 3. As well, trehalose is used as a sweetener in the food industry, and it is extensively used for product preservation in both the food and cosmetics industries. The adoption of trehalose for these types of commercial applications was initially limited by the inability to obtain bulk quantities of pure trehalose from natural sources or through synthesis. However, an efficient enzymatic process for the economical production of trehalose from starch has recently been developed, which has spurred its widespread commercial use 5.
Chemically modified derivatives of trehalose, referred to herein as trehalose analogues, have gained increasing attention for various applications (generic structure shown in Figure 1A; specific examples of trehalose analogues shown in Figure 1B) 6. For example, lacto-trehalose is a trehalose analogue with one of its glucose units replaced with galactose, thus its 4-position hydroxyl group has an inverted stereochemical configuration. Lacto-trehalose has the same stabilizing properties as trehalose but is resistant to degradation by intestinal enzymes, making it attractive as a non-caloric food additive 6, 7.
Our group&#39;s interest in trehalose analogues primarily relates to their value as mycobacteria-specific probes and inhibitors. The Barry and Davis groups developed a fluorescein-conjugated keto-trehalose analogue, named FITC-keto-trehalose, which was shown to metabolically label the cell wall of live M. tuberculosis, enabling its detection by fluorescence microscopy 8. The Bertozzi lab developed smaller azido-trehalose (TreAz) analogues that could metabolically label the cell wall and subsequently be detected using click chemistry and fluorescence analysis 9. These advances point to the possibility of using trehalose-based probes as diagnostic imaging agents for tuberculosis. Trehalose analogues have also been pursued as inhibitors of M. tuberculosis due to their potential to disrupt pathways in the bacterium that are essential for viability and virulence 10,11,12.
So far, the main obstacle to developing trehalose analogues for bio(techno)logical and biomedical applications is the lack of efficient synthetic methods. The two traditional routes to producing trehalose analogues rely on chemical synthesis (Figure 2). One route involves desymmetrization/modification of natural trehalose, while the other involves starting with properly functionalized monosaccharide building blocks and performing chemical glycosylation to forge the 1,1-&#945;,&#945;-glycosidic bond. These approaches, which have recently been discussed in review articles 13, 14, have proven useful for accomplishing multistep synthesis of small quantities of complex trehalose-containing natural products, such as sulfolipid-1 from M. tuberculosis 15. However, both approaches are generally inefficient, time-consuming, inaccessible to non-chemists, and, additionally, are not considered to be environmentally friendly. Thus, for synthesizing certain types of trehalose analogues, these strategies are not ideal.
<img alt="Figure 2" src="/files/ftp_upload/54485/54485fig2.jpg" /> 
Figure 2: Approaches to trehalose analogue synthesis. Chemical approaches to trehalose analogue synthesis, shown on the left, use multistep procedures that involve difficult protection/deprotection, desymmetrization, and/or glycosylation steps. Enzymatic synthesis, shown on the right, uses enzyme(s) to stereoselectively convert simple, unprotected substrates to trehalose analogues in aqueous solution. The enzymatic protocol reported herein uses a trehalose synthase (TreT) enzyme to convert glucose analogues and UDP-glucose into trehalose analogues in a single step. <a href="http://cloudflare.jove.com/files/ftp_upload/54485/54485fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
An efficient biocatalytic route to trehalose analogues would facilitate the production, evaluation, and application of this promising class of molecules. While the commercial enzymatic process for trehalose production5 is not adaptable to synthesizing analogues because it utilizes starch as a substrate, there are other biosynthetic pathways in nature that may be exploited for trehalose analogue synthesis. However, research in this area, which was recently reviewed 6, has been limited. One report used a method inspired by the Escherichia coli trehalose biosynthetic pathway to access a single fluoro-trehalose analogue from the corresponding fluoro-glucose. However, this approach requires a three-enzyme system that has limited efficiency and generality 8. Another approach that has been explored is to use trehalose phosphorylase (TreP) in the reverse direction, which in principle permits the one-step synthesis of trehalose analogues from glucose analogues and glucose-1-phosphate 6, 16, 17. Although this approach may have future promise, both inverting and retaining TrePs currently have drawbacks for analogue synthesis. For example, inverting TrePs have a prohibitively expensive donor molecule (&#946;-D-glucose 1-phosphate) and retaining TrePs have poor enzyme expression yields/stability and limited substrate promiscuity. Significant improvements (e.g., via enzyme engineering) will be needed before TreP-mediated analogue synthesis is practical.
At present, the most practical approach for the enzymatic synthesis of trehalose analogues is to use a trehalose synthase (TreT) enzyme, which converts glucose and uridine diphosphate (UDP)-glucose into trehalose in a single step 6. We recently reported the use of Thermoproteus tenax TreT&#8212;a thermostable and unidirectional enzyme 18&#8212;to synthesize trehalose analogues from glucose analogues and UDP-glucose (Figure 3) 19. This enzyme only operates in the synthetic direction and avoids the problem of trehalose degradation found in the TreP system. This one-step reaction could be completed in 1 hour, and a broad variety of trehalose analogues were accessed in high yield (up to &#62;99% as determined by high performance liquid chromatography (HPLC)) from readily available glucose analogue substrates (see Table 1 in the Representative Results section).
<img alt="Figure 3" src="/files/ftp_upload/54485/54485fig3.jpg" /> 
Figure 3: TreT-catalyzed one-step synthesis of trehalose analogues. The TreT enzyme from T. tenax can stereoselectively join readily available glucose analogues and UDP-glucose to form trehalose analogues in one step. R1-R4 = Variable structural modification, for example azido-, fluoro-, deoxy-, thio-, stereochemical, or isotopic label modifications; Y = variable heteroatom, for example oxygen or sulfur, or isotopically labeled heteroatom.
Here, we provide a detailed protocol for the TreT synthesis process, including expression and purification of TreT from E. coli, optimized TreT reaction conditions, and an improved purification method that is carried out entirely in the aqueous phase. This modified protocol enables the expedient and efficient synthesis and purification of diverse trehalose analogues on a semi-preparative scale (10-100 mg). We also demonstrate the use of this protocol for preparing and administering a trehalose-based probe to mycobacteria in less than 1 hour, which enabled the rapid fluorescence detection of mycobacterial cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LB agar</td>
			<td>Research Products International</td>
			<td>L24021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria broth</td>
			<td>Research Products International</td>
			<td>L24045</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth</td>
			<td>Research Products International</td>
			<td>T15050</td>
			<td></td>
		</tr>
		<tr>
			<td>L-(+)-Arabinose</td>
			<td>Sigma Aldrich</td>
			<td>A3256</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline</td>
			<td>GE Healthcare</td>
			<td>SH30256</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BDH</td>
			<td>BDH9286</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate,</td>
			<td rowspan="2">Fisher Scientific</td>
			<td rowspan="2">S374</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>monobasic</td>
		</tr>
		<tr>
			<td>Syringe filter, 0.45 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>09719D</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor mini-tablets, EDTA-free</td>
			<td>Thermo Scientific</td>
			<td>88666</td>
			<td></td>
		</tr>
		<tr>
			<td>HisTrap HP nickel affinity column, 5 mL</td>
			<td>GE Healthcare</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS base ultrapure</td>
			<td>Research Products International</td>
			<td>T60040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing, MWCO 12&#8211;14,000</td>
			<td>Fisher Scientific</td>
			<td>21-152-16</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Glucose analogues</td>
			<td>CarboSynth,</td>
			<td rowspan="3"></td>
			<td rowspan="3">Examples of vendors that offer numerous glucose analogues</td>
		</tr>
		<tr>
			<td>Sigma Aldrich,</td>
		</tr>
		<tr>
			<td>Santa Cruz Biotechnology, American Radiolabeled Chemicals</td>
		</tr>
		<tr>
			<td>6-Azido-6-deoxy glucopyranose (6-GlcAz)</td>
			<td>CarboSynth</td>
			<td>MA02620</td>
			<td></td>
		</tr>
		<tr>
			<td>UDP-Glucose</td>
			<td>abcam Biochemicals</td>
			<td>ab120384</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate&#160;</td>
			<td>Fisher Scientific</td>
			<td>M33</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 centrifugal filter unit</td>
			<td>EMD Millipore</td>
			<td>UFC901008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rex RG 501-X8 mixed-bed ion-exchange resin</td>
			<td>Bio-Rad</td>
			<td>444-9999</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra-Fine Bio-Gel P2 media</td>
			<td>Bio-Rad</td>
			<td>150-4118</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-backed silica gel thin-layer chromatography plates</td>
			<td>EMD Millipore</td>
			<td>1056280001</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Butanol</td>
			<td>Fisher Scientific</td>
			<td>A399</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>S25310A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Fisher Scientific</td>
			<td>A300</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>EMD Millipore</td>
			<td>AX0145</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide, 99.8%</td>
			<td>Acros Organics</td>
			<td>351430075</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminopropyl HPLC column</td>
			<td>Sigma Aldrich</td>
			<td>58338</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Para-formaldehyde</td>
			<td>Ted Pella</td>
			<td>18505</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkyne-488</td>
			<td>Sigma Aldrich</td>
			<td>761621</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td>Click Chemistry Tools</td>
			<td>1061</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butanol</td>
			<td>Sigma Aldrich</td>
			<td>360538</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>W387520</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) sulfate</td>
			<td>Sigma Aldrich</td>
			<td>C1297</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting medium</td>
			<td>Southern Biotechnology</td>
			<td>10001</td>
			<td></td>
		</tr>
	</tbody>
</table>

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).

T. tenax TreT was obtained from E. coli in a yield of approximately 4 mg/L using standard protein expression and purification techniques. A single nickel affinity chromatography step was sufficient to purify TreT from E. coli lysate (a representative FPLC trace is shown in Figure 4). As established in our initial publication on the TreT synthesis process, recombinant T. tenax TreT is capable of converting a broad of variety glucose anal...

Watch this Scientific Journal Video about Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues at JoVE.com

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

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 ...

The overall goal of this method is to rapidly synthesize and purify trehalose analogues which have possible applications ranging from the detection of pathogenic bacteria to the preservation of bio materials. This is a fast and high-yielding method that provides access to analogues of the disaccharide trehalose. These compounds have various established and emerging applications in biomedical and biotechnological research.This method consists of a one-step enzymatic synthesis and an all-aqueous purification. Together, these procedures help to address some of the drawbacks of traditional chemical syntheses of trehalose analogues which can be lengthy, low yielding and environmentally unfavorable. The implications of this method may extend toward the study and diagnosis of tuberculosis because modified trehalose analogues have been used for imaging microbacterium tuberculosis, the causative agent of tuberculosis.This method also can help accelerate the synthesis and evaluation of trehalose analogues for other possible applications such as preservation of biological products and use as noncaloric sweeteners. Prior to beginning the procedure, prepare an overnight culture of TreT-expressing E.coli. To begin the TreT expression procedure, place 750 milliliters of TB growth medium in a 2, 800 milliliter Fernbach culture flask and autoclave it.Add ampicillin to the broth to achieve a final concentration of 100 micrograms per milliliter. Obtain cuvettes for UV-vis spectroscopy and fill one cuvette with broth from the flask as a blank. Next, add three milliliters of the prepared TreT-expressing E.coli overnight culture to the flask.Incubate the flask at 37 degrees Celsius while shaking at 200 rpm. Check the absorbance of the broth at 600 nanometers against the broth blank periodically. When the absorbance value at 600 nanometers is between 0.5 and 1.0, add 750 microliters of arabinose to the flask to induce TreT expression.Incubate the culture at 37 degrees Celsius overnight while shaking at 200 rpm. Next, place the culture in a one liter polypropylene centrifuge bottle and centrifuge the culture at 4, 000 times G for 15 minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet in 15 milliliters phosphate buffered saline.Transfer the suspension to a conical centrifuge tube and centrifuge under the same conditions. Then discard the supernatant and if necessary, store the pellet at minus 80 degrees Celsius. Dissolve a protease inhibitor mini tablet in 20 milliliters of wash buffer.Add the protease inhibitor solution to the pellet and vortex the mixture to resuspend the pellet. Transfer the suspension to a 100 milliliter beaker. Next, sonicate the suspension to lyse the cells positioning the probe about one centimeter from the bottom of the beaker.Place the lysate in a metal conical tube and centrifuge the lysate at 15, 000 times G at four degrees Celsius for 60 minutes. Filter the lysate with a syringe filter. Isolate TreT by fast protein liquid chromatography and assess the purity with SDS page.Then exchange TreT into Tris buffer by dialysis overnight at four degrees Celsius to form the TreT solution for trehalose analogue synthesis. Collect the blank from the dialysis washing. Determine the TreT concentration by UV-vis spectroscopy against the dialysis blank.To begin synthesis of 6-Azido-trehalose using TreT on the 20 to 30 milligram scale, place 0.08 millimole 6-Azido-glucose, 0.16 millimole UDP-glucose and 0.08 millimole magnesium dichloride in a 15 millimeter conical tube. Add the TreT and Tris buffer. Dilute the mixture with Tris buffer as needed to achieve an enzyme concentration of 300 micrograms per milliliter in a final volume of four milliliters.Cap and invert the tube to dissolve the solids. Incubate the reaction mixture for one hour at 70 degrees Celsius while shaking at 300 rpm. During the incubation, place three milliliters deionized water into a 10 kilodalton centrifugal filter unit and centrifuge at 3, 000 times G until all liquid is in the tube.Repeat the wash twice more to ensure thorough rinsing of the filter unit. When the reaction is complete, cool the mixture to room temperature on ice. Then transfer the product mixture to the centrifugal filter unit.Rinse the reaction tube with one milliliter deionized water three times and transfer each rinse to the centrifugal filter unit. Centrifuge the mixture at 3, 000 times G until all liquid has passed into the tube. Add three milliliters deionized water to the filter unit and centrifuge again under the same conditions.Repeat the rinse as needed to maximize product recovery. Add three grams mixed bed ion exchange resin to the filtrate. Stir the mixture with a stir bar at room temperature for one hour.After stirring, decant and filter the supernatant. Rinse the resin with five milliliters deionized water and filter the rinse. Repeat as needed to maximize product recovery and combine all filtrates.Concentrate the combined filtrate. Then to check for unreacted 6-GlcAz precursor, prepare silica gel TLC plate with spots of 6-GlcAz, 6-TreAz, the concentrated product and a co-spot. Develop the plate with a mixture of n-butanol, ethanol and deionized water.Once the plate is dry, visualize the spots by dipping the plate in 5%sulfuric acid and ethanol and heating the plate on a hot plate on high setting for about five minutes. If unreacted precursor is observed, purify the product mixture on a size exclusion column and repeat the TLC analysis. Once the pure trehalose analogue has been obtained, remove water to obtain the dried 6-TreAz product.The TreT enzyme stereo selectively joins the glucose analogue and UDP-glucose to form the corresponding trehalose analogue. The reaction scope include stereo chemical deoxifluoro, thio and azido modifications. However, substituents at the four position on the ring are less well-tolerated.Using this procedure, the glucose analogue 6-GlcAz was quantitatively converted to the trehalose analogue 6-TreAz in less than 60 minutes as confirmed by TLC. The pure product was isolated with the non-chromatographic spin dialysis ion exchange purification method and characterized by NMR in HPLC. The pure 6-TreAz solution was then used to perform azide labeling of the model bacterium M.smegmatis.Subsequent fluorophore labeling by a click chemistry reaction with the cell surface azides showed that M.smegmatis with a functional trehalose transporter had been successfully labeled with azides whereas the strain without was not. Once mastered, the TreT catalyze reaction and subsequent purification can be performed in one to four hours depending on the number of rinse steps used to maximize production recovery. While attempting this procedure, it's important to remember that the reaction product should always be analyzed by NMR and TLC or HPLC to confirm its purity and structure.Following this procedure, the synthesized trehalose analogue can be evaluated or employed as a bacterial imaging probe, metabolic tracer, enzyme inhibitor or biomolecule stabilizing agent. This method provides a way for researchers to prepare trehalose analogues with modifications such as stable isotope or radioisotope labels. Altogether, the compounds accessible through this method should be useful in tuberculosis research and other biomedical research areas.After watching this video, you should have a good understanding of how to express and purify the TreT enzyme, use TreT to synthesize trehalose analogues in one step and purify the trehalose analogue products.

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Research

JoVE Journal

Biochemistry

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

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.

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 ...

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 ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...