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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose:+Current+use+and+future+applications.">Trehalose: Current use and future applications.</a> J. Pharm. Sci. 100, 2020-2053 (2011).</li><li>Adams, R. P., Kendall, E., Kartha, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+free+sugars+in+growing+and+desiccated+plants+of+Selaginella+lepidophylla.">Comparison of free sugars in growing and desiccated plants of Selaginella lepidophylla.</a> Biochem. Syst. 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. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+synthesis+of+a+galactose-containing+trehalose+analogue+disaccharide+by+Pyrococcus+horikoshii+trehalose-synthesizing+glycosyltransferase:+Inhibitory+effects+on+several+disaccharidase+activities.">Enzymatic synthesis of a galactose-containing trehalose analogue disaccharide by Pyrococcus horikoshii trehalose-synthesizing glycosyltransferase: Inhibitory effects on several disaccharidase activities.</a> J. Mol. Catal. B: Enzym. 49, 98-103 (2007).</li><li>Backus, K. 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=Uptake+of+unnatural+trehalose+analogs+as+a+reporter+for+Mycobacterium+tuberculosis.">Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis.</a> Nat. Chem. Biol. 7, 228-235 (2011).</li><li>Swarts, B. 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 ...

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