Name: thermostabilization, expression, purification, and crystallization of the human serotonin transporter bound to s-citalopram
Uploaded: 2016-11-27T15:00:00.000+00:00
Duration: 12 min 21 s
Description: Watch this Scientific Journal Video about Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram at JoVE.com

We thank D. Cawley for generating monoclonal antibodies. We thank A. Penmatsa and K. Wang for sharing ideas and expertise developed from the dopamine transporter. L. Vaskalis for assistance with figures, H. Owen for help with manuscript preparation and other Gouaux laboratory members for helpful discussions. J.A.C. has support from a Banting postdoctoral fellowship from the Canadian Institutes of Health Research. E.M.G. is supported by the National Science Foundation Graduate Research Fellowship. We are particularly grateful to Bernie and Jennifer LaCroute for their generous support, as well as for funding from the NIH (5R37MH070039). E.G. is an investigator of the Howard Hughes Medical Institute.

<ol>
	<li>Kristensen, A. 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=SLC6+neurotransmitter+transporters:+structure,+function,+and+regulation.">SLC6 neurotransmitter transporters: structure, function, and regulation.</a> Pharmacol Rev. 63 (3), 585-640 (2011).</li><li>Broer, S., Gether, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+solute+carrier+6+family+of+transporters.">The solute carrier 6 family of transporters.</a> Br J Pharmacol. 167 (2), 256-278 (2012).</li><li>Andersen, J., Kristensen, A. S., Bang-Andersen, B., Stromgaard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+understanding+of+the+interaction+of+antidepressant+drugs+with+serotonin+and+norepinephrine+transporters.">Recent advances in the understanding of the interaction of antidepressant drugs with serotonin and norepinephrine transporters.</a> Chem Commun (Camb). 25 (25), 3677-3692 (2009).</li><li>Hahn, M. K., Blakely, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+impact+of+SLC6+transporter+genetic+variation.">The functional impact of SLC6 transporter genetic variation.</a> Annu Rev Pharmacol Toxicol. 47, 401-441 (2007).</li><li>Abdul-Hussein, S., Andrell, J., Tate, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermostabilisation+of+the+serotonin+transporter+in+a+cocaine-bound+conformation.">Thermostabilisation of the serotonin transporter in a cocaine-bound conformation.</a> J Mol Biol. 425 (12), 2198-2207 (2013).</li><li>Green, E. M., Coleman, J. A., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermostabilization+of+the+human+serotonin+transporter+in+an+antidepressant-Bound+Conformation.">Thermostabilization of the human serotonin transporter in an antidepressant-Bound Conformation.</a> Plos One. 10 (12), e0145688 (2015).</li><li>Goehring, A., 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=Screening+and+large-scale+expression+of+membrane+proteins+in+mammalian+cells+for+structural+studies.">Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.</a> Nat Protoc. 9 (11), 2574-2585 (2014).</li><li>Kawate, T., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-detection+size-exclusion+chromatography+for+precrystallization+screening+of+integral+membrane+proteins.">Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins.</a> Structure. 14 (4), 673-681 (2006).</li><li>De Becker, G., 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=The+adjuvant+monophosphoryl+lipid+A+increases+the+function+of+antigen-presenting+cells.">The adjuvant monophosphoryl lipid A increases the function of antigen-presenting cells.</a> Int Immunol. 12 (6), 807-815 (2000).</li><li>Coleman, J. A., Green, E. M., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+structures+and+mechanism+of+the+human+serotonin+transporter.">X-ray structures and mechanism of the human serotonin transporter.</a> Nature. , (2016).</li><li>White, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progress+of+membrane+protein+structure+determination.">The progress of membrane protein structure determination.</a> Protein Sci. 13 (7), 1948-1949 (2004).</li><li>Gambotto, A., 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=Immunogenicity+of+enhanced+green+fluorescent+protein+(EGFP)+in+BALB/c+mice:+identification+of+an+H2-Kd-restricted+CTL+epitope.">Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope.</a> Gene Ther. 7 (23), 2036-2040 (2000).</li><li>Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W., Howard, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+major+histocompatibility+antigens+produced+by+hybrid+cell+lines.">Antibodies to major histocompatibility antigens produced by hybrid cell lines.</a> Nature. 266 (5602), 550-552 (1977).</li><li>Deller, M. C., Kong, L., Rupp, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+stability:+a+crystallographer's+perspective.">Protein stability: a crystallographer's perspective.</a> Acta Crystallogr F Struct Biol Commun. 72, 72-95 (2016).</li><li>Hattori, M., Hibbs, R. E., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-detection+size-exclusion+chromatography-based+thermostability+assay+for+membrane+protein+precrystallization+screening.">A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening.</a> Structure. 20 (8), 1293-1299 (2012).</li><li>Tate, C. G., 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=Comparison+of+seven+different+heterologous+protein+expression+systems+for+the+production+of+the+serotonin+transporter.">Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter.</a> Biochim Biophys Acta. 1610 (1), 141-153 (2003).</li><li>Rigaud, J. L., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+liposomes.">Reconstitution of membrane proteins into liposomes.</a> Methods Enzymol. 372, 65-86 (2003).</li><li>Caffrey, M., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallizing+membrane+proteins+using+lipidic+mesophases.">Crystallizing membrane proteins using lipidic mesophases.</a> Nat Protoc. 4 (5), 706-731 (2009).</li><li>Faham, S., Bowie, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicelle+crystallization:+a+new+method+for+crystallizing+membrane+proteins+yields+a+monomeric+bacteriorhodopsin+structure.">Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure.</a> J Mol Biol. 316 (1), 1-6 (2002).</li></ol>

The authors have nothing to disclose.

Determination of membrane protein structure by biophysical techniques remains a daunting undertaking for many medically significant transporters, receptors, and channels11. Here we share detailed expertise developed for the structure determination of the human serotonin transporter bound to S-citalopram. We anticipate that these methods will be useful to determine structures of SERT in other conformational states as well as structures of other difficult membrane proteins. Furthermore, the biochemical techniques described here can also be used to study function of purified SERT in detergent and a near-native lipid environment.
SERT crystallization hinged upon the development of several tools and techniques. First, improvements in transporter thermostability produced SERT variants which were well-behaved in various detergent micelles following extraction of the transporter from membranes6. Second, the use of the high-affinity ligand S-citalopram throughout purification and crystallization further improved stability and reduced conformational heterogeneity. Third, the development of the BacMam expression system7 allowed for production of large amounts of SERT in a short period of 2 weeks, facilitating both immunization and crystallization. Finally, the development of strategies to select for high-affinity antibodies which recognize 3D epitopes allowed for the discovery of the 8B6 antibody which promotes well-ordered packing of SERT-antibody complexes into crystals.
There are a number of critical steps and reagents as well as common problems which often occur throughout the protocol. First, the generation of high titer SERT P2 virus can be problematic. Adding low concentrations of P1 virus to generate P2 virus as described in this protocol usually mitigates this problem, and in cases where the P2 virus titer is low, P3 virus can be made using virus at an MOI of 0.0001. Viruses with a titer less than 1 x 108 virus particles/mL&#160;should not be used and will almost always result in low protein yields. For expression, HEK293S GnTI- cells were chosen since they lack N-acetylglucosaminyltransferase I activity and thus cannot synthesize complex N-glycans, instead producing only high mannose N-glycans. EndoH cleaves N-linked glycosylation of high mannose glycans at two sites in extracellular loop 2 (EL2), leaving an N-acetylglucosamine attached to asparagine. Digestion of N-linked sugars reduces surface entropy of EL2 which is likely important for crystallization. For the generation of antibodies, the SERTIC should be used for immunization. GFP is highly immunogenic12 and should not be used as a fusion tag to generate antibodies since it is difficult to completely remove by SEC. The flexible N- and C-terminus of SERT were also not included in the construct in order to avoid antibodies against these regions. Mice can be immunized with 30 &#181;g of reconstituted protein; continue immunizing mice until high serum concentrations of antibodies can be detected and produce hybridoma cells as described13. A thermostabilized construct is usually the best choice for immunization; if the transporter is well behaved and retains biological activity following purification, this is often sufficient to raise antibodies. The 8B6 antibody was raised against WT SERT. For crystallization, only the peak fractions from SEC containing monodisperse complex as judged by FSEC should be combined and concentrated. SERT-8B6 crystals grow in a narrow range of conditions and there are a number of steps which should be taken to troubleshoot problems specifically related to SERT crystal growth. Tris base adjusted with HCl should not be used in the reservoir solution, since this buffer does not support crystal growth; it is thus critical to instead use Tris adjusted with NaOH. SERT crystals grow in a narrow concentration range of PEG 400, so if crystals do not grow or if many small crystals are observed, even a small increase or decrease in the PEG 400 concentration would be advised. Furthermore, the additive 6-aminohexanoic acid was also used in the optimized screen to improve nucleation. The drop ratio of protein:well solution is also a key determining factor for crystal growth. Drop ratios of 1.5 - 2:1 are recommended, with drop ratios closer to 2:1 typically supporting the growth of larger 3-dimensional crystals. Finally, the use of low profile 24-well plates is also crucial toward crystal growth, presumably due to modification of the rate of vapor diffusion.
An alternative approach to the SPA method has been developed to screen for mutants which stabilize the rat serotonin transporter in a cocaine-bound conformation using a filter binding assay5. By contrast, the SPA based assay allows for sequential heating steps following by determination of the fraction of SERT which remains bound to ligand. Thus, this allows for the rapid determination of the melting temperature from a small number of samples. The SPA method relies on the availability of a radiolabeled high-affinity ligand and if no ligands are known which bind with submicromolar affinity then an alternative approach will be necessary. Many other methods are commonly used to measure protein stability such as binding of fluorescent dyes and calorimetry14 but are low-throughput and are either unable to directly measure function or require large amounts of protein. If the SPA method cannot be used, one alternative high-throughput approach is an FSEC-based Thermostability Assay15 (FSEC-TS), where the sample is heated followed by separation of the fraction of remaining transporter. FSEC-TS is a useful approach for accessing chromatographic behavior and oligomeric state and is a powerful complementary tool which can be used alongside the SPA method.
A comparison of various common protein expression systems was also found to favor the use of mammalian cells for SERT expression16 and unsurprisingly this is likely the case for many proteins of mammalian origin. The methods which we have used for expression have been tailored for SERT but are likely readily adaptable. Conditions which favor high levels of expression should be carefully identified by varying the time of expression, temperature, virus concentration, and presence of histone deacetylase inhibitors such as sodium butyrate.
We generally favor affinity purification in a mild long-chain detergent such as C12M together with CHS prior to reconstitution to retain high-affinity ligand binding. Reconstitution using hydrophobic absorption is a mild technique which we have found to be effective for several other transporters and receptors. If this is not successful, removal of detergents with high critical micelle concentrations by dialysis, dilution, or SEC can be employed17 provided the antigen is sufficiently stable in such detergents. In cases where no suitable antibodies are found, we almost always find the issue is due to loss of function or denaturation of the antigen and in such cases we successfully performed new immunizations paying special attention to protein biochemistry. Finally, ligands and antibodies which bind with high-affinity should form the basis for a rationally planned crystallization experiment and by taking advantage of a thermostable variant, one can screen a broader range of conditions by varying the properties of different detergents. Furthermore, crystallization in a lipidic mesophase18 or using bicelles19 should always be considered as an alternative to crystallization in micelles.
These principals and methods can be used with some modification for many other transmembrane proteins which are difficult to express and purify from other expression hosts, and will be particularly useful for the structure determination of targets of high-affinity drugs.

The serotonin transporter (SERT) is an integral membrane protein that facilitates the transport of serotonin across cellular membranes1. SERT belongs to a family of Neurotransmitter Sodium Symporters (NSSs) which also includes the dopamine and norepinephrine transporters2. SERT is the molecular target of widely prescribed antidepressant and anti-anxiety drugs which act to competitively inhibit serotonin transport3. SERT exploits the energetically favorable cotransport of sodium to remove neurotransmitter from the synaptic cleft. Extensive characterization of the serotonergic system has shown that changes in serotonin metabolism appear to influence virtually all neurological processes including mood, sleep, pain, cognition, and aggression behaviors4. SERT function can be modified through the use of antidepressants and Selective Serotonin Reuptake Inhibitors (SSRIs) like S-citalopram, as well as by psychostimulants and drugs of addiction such as amphetamine and 3,4-methylenedioxy-N-methylamphetamine or &#34;ecstasy&#34;1,2.
SSRIs are tremendously important for the treatment of mood disorders, yet the precise structural basis for their action is not well understood. WT SERT is unstable in detergent micelles, thus impeding progress towards a three-dimensional (3D) structure of SERT5,6. Recently, we developed variants of SERT which are robustly stable in a wide range of detergents and retain SSRI binding activity6. These thermostable SERT variants were selected using a scintillation proximity-based thermostability assay. Here we describe a procedure for the generation of high-affinity antibodies which can bind SERT, and the purification and crystallization of thermostable SERT, in complex with antibody and S-citalopram.
This protocol assumes that the SERT and 8B6 genes have been successfully cloned into the BacMam7 and insect expression vectors, respectively. To generate antibodies, cDNA encoding residues 73-616 of WT SERT was cloned into the BacMam vector with a C-terminal Strep II tag (SERTIC). For the thermostability screen, SERT residues 73-616 with C-terminal GFP, Strep II, and 10-His tags (SERTTC) was used. Individual point mutations were generated in the SERTTC background. For the crystallization protocol, the SERT-GFP fusion protein was used with Twin-Strep [TrpSerHisProGlnPheGluLys(GlyGlyGlySer)2GlyGlySerAlaTrpSerHisProGlnPheGluLys] and 10-His tags, carrying the thermostable mutants, Y110A, I291A, and T439S and mutations of surface cysteines C554A, C580A, and C622A (SERTCC). Thrombin cleavage sequences (LVPRGS) were also inserted in the SERTCC after Q76 and T618 to allow removal of the N- and C-termini. The plasmid encoding the 8B6 Fab was engineered to express both the heavy and light chains of the antibody with GP67 secretion sequences under the control of two separate polyhedrin promoters. The C-terminus of the heavy chain of the 8B6 antibody was tagged with an 8-His tag and a thrombin cleavage site was inserted between the heavy chain and the tag.

<table><tbody><tr><td>DH10Bac</td><td>Invitrogen</td><td>10361-012</td><td></td></tr><tr><td>Kanamycin</td><td>Fisher</td><td>BP906-5</td><td></td></tr><tr><td>Gentamicin</td><td>Fisher</td><td>BP918-1</td><td></td></tr><tr><td>Tetracycline</td><td>Sigma</td><td>T-7660</td><td></td></tr><tr><td>Bluo-gal</td><td>Invitrogen</td><td>15519-028</td><td>5-Bromo-3-indolyl &amp;beta;-D-galactopyranoside</td></tr><tr><td>Isopropyl beta-D-1-thiogalactopyranoside</td><td>Anatrace</td><td>I1003</td><td>IPTG</td></tr><tr><td>Miniprep kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>Cellfectin II</td><td>Invitrogen</td><td>10362-100</td><td>Sf9 transfection reagent</td></tr><tr><td>Sf9</td><td>ATCC</td><td>CRL-1711</td><td></td></tr><tr><td>Sf-900 III SFM media</td><td>Life Technologies</td><td>12658-027</td><td></td></tr><tr><td>HEK-293S GnTI&lt;sup&gt;-&lt;/sup&gt;</td><td>ATCC</td><td>CRL-3022</td><td></td></tr><tr><td>Freestyle 293 media</td><td>Life Technologies</td><td>12338-018</td><td>293 expression media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>0984018DJ</td><td></td></tr><tr><td>Sodium butyrate</td><td>Sigma</td><td>303410</td><td></td></tr><tr><td>&lt;em&gt;S&lt;/em&gt;-citalopram</td><td>Sigma</td><td>E4786</td><td>Anagrade</td></tr><tr><td>n-Dodecyl-beta-D-Maltopyranoside</td><td>Anatrace</td><td>D310</td><td></td></tr><tr><td>Cholesteryl hemmisuccinate</td><td>Sigma</td><td>C6013</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>850457P</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine</td><td>Avanti Polar Lipids</td><td>850757P</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoglycerol</td><td>Avanti Polar Lipids</td><td>840457P</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884</td><td></td></tr><tr><td>Pepstatin A</td><td>Sigma</td><td>P5318</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Desthiobiotin</td><td>Iba Life Sciences</td><td>2-1000-05</td><td></td></tr><tr><td>Asolectin</td><td>Sigma</td><td>11145</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C-8667</td><td></td></tr><tr><td>Lipid A</td><td>Sigma</td><td>L5399</td><td></td></tr><tr><td>Brain polar lipid</td><td>Avanti Polar Lipids</td><td>141101C</td><td></td></tr><tr><td>Biobeads</td><td>Biorad</td><td>152-3920</td><td>Hydrophobic absorption resin</td></tr><tr><td>Goat anti-mouse IRDye 680RD</td><td>Odyssey</td><td>926-68070</td><td>Used as secondary antibody for western blotting</td></tr><tr><td>Lauryl maltose neopentyl glycol</td><td>Anatrace</td><td>NG310</td><td>Anagrade</td></tr><tr><td>Serotonin</td><td>Sigma</td><td>H9523</td><td></td></tr><tr><td>pFastBac 8B6</td><td></td><td></td><td>Available from authors</td></tr><tr><td>pEG Bacmam SERT Strep II</td><td></td><td></td><td>SERT&lt;sub&gt;IC&lt;/sub&gt;, Available from authors</td></tr><tr><td>pEG Bacmam SERT GFP twin Strep His</td><td></td><td></td><td>SERT&lt;sub&gt;TC&lt;/sub&gt;, Available from authors</td></tr><tr><td>pEG Bacmam SERT ts3 GFP twin Strep His</td><td></td><td></td><td>SERT&lt;sub&gt;CC&lt;/sub&gt;, Available from authors</td></tr><tr><td>Imidazole</td><td>Sigma</td><td>56749</td><td></td></tr><tr><td>n-Octyl &amp;beta;-D-maltoside</td><td>Anatrace</td><td>O310</td><td>Anagrade</td></tr><tr><td>Thrombin</td><td>Haematologic Technologies</td><td>HCT-0020</td><td></td></tr><tr><td>EndoH</td><td>New England Biolabs</td><td>P0702</td><td></td></tr><tr><td>Trizma-HCl</td><td>Sigma</td><td>T5941</td><td>Tris is used for preparation of crystallization reservoirs</td></tr><tr><td>PEG 400</td><td>Sigma</td><td>91893</td><td></td></tr><tr><td>6-aminohexanoic acid</td><td>Sigma</td><td>7260</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Fisher</td><td>MT25052CV</td><td></td></tr><tr><td>Isoplate-96 TC</td><td>PerkinElmer</td><td>6005070</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen</td><td>SL100688</td><td>Polymer transfection reagent for mamalian cells</td></tr><tr><td>Copper HIS-Tag YSI SPA Beads</td><td>PerkinElmer</td><td>RPNQ0096</td><td>His-tag affinity SPA beads</td></tr><tr><td>Citalopram, [N-Methyl-&lt;sup&gt;3&lt;/sup&gt;H]</td><td>PerkinElmer</td><td>NET1039250UC</td><td></td></tr><tr><td>ThermoMixer C</td><td>Eppendorf</td><td>5382000023</td><td>heating block for thermostability assay</td></tr><tr><td>ThermoTop</td><td>Eppendorf</td><td>5308000003</td><td></td></tr><tr><td>SmartBlock PCR 96, thermoblock for PCR plates</td><td>Eppendorf</td><td>5306000006</td><td></td></tr><tr><td>0.2 &amp;micro;m syringe filter</td><td>Olympus Plastics</td><td>25-243</td><td></td></tr><tr><td>1 L filter system</td><td>Corning</td><td>430517</td><td></td></tr><tr><td>2 L flat bottom tissue culture flask</td><td>Genemate</td><td>F-5909-2000</td><td></td></tr><tr><td>2 L baffled tissue culture flask</td><td>Genemate</td><td>F-5909-2000B</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Scientific</td><td>3950</td><td></td></tr><tr><td>Forma Orbital Shaker</td><td>Thermo Scientific</td><td>416</td><td></td></tr><tr><td>Strep-Tactin resin</td><td>Iba Life Sciences</td><td>2-1208-025</td><td>Strep affinity resin</td></tr><tr><td>Extruder</td><td>Northern Lipids</td><td></td><td></td></tr><tr><td>Li-Cor imaging system</td><td>Odyssey</td><td></td><td>western blot imaging system</td></tr><tr><td>XK16 column</td><td>GE Healthcare</td><td>28-9889-37</td><td>column used for Strep-Tactin and Talon purificaiton</td></tr><tr><td>100 kDa MWCO protein concentrator</td><td>Millipore</td><td>UFC910096</td><td></td></tr><tr><td>30 kDa MWCO protein concentrator</td><td>Millipore</td><td>UFC903024</td><td></td></tr><tr><td>&amp;Auml;kta FPLC</td><td>GE Healthcare</td><td>UPC-900</td><td></td></tr><tr><td>HPLC</td><td>Shimadzu</td><td>51476</td><td></td></tr><tr><td>Superose 6 (10/300) column</td><td>GE Healthcare</td><td>17-5172-01</td><td>Used for FSEC</td></tr><tr><td>Tangential flow apparatus</td><td>Pall Filtron</td><td></td><td></td></tr><tr><td>0.2 &amp;micro;m filter tangential flow cell</td><td>Pall Filtron</td><td>PSM20C11</td><td></td></tr><tr><td>30 kDa MWCO tangential flow concentrator</td><td>Pall Filtron</td><td>OS030T12</td><td></td></tr><tr><td>Talon resin</td><td>Clonetech</td><td>635504</td><td>His-tag affinity resin used for Fab purification</td></tr><tr><td>1 mL HiTrap SP column</td><td>GE Healthcare</td><td>17115101</td><td>Cation exchanger used for Fab purification</td></tr><tr><td>Superdex 200 10/300 GL column</td><td>GE Healthcare</td><td>17-5175-01</td><td>Used for SEC separation of SERT-8B6</td></tr><tr><td>24-well VDXm plate</td><td>Hampton Research</td><td>HR3-306</td><td></td></tr><tr><td>18 mm coverslips</td><td>Hampton Research</td><td>HR3-239</td><td></td></tr><tr><td>Virocyt virus counter</td><td>Virocyt</td><td>2100</td><td></td></tr><tr><td>MicroBeta Trilux</td><td>PerkinElmer</td><td>1450</td><td>96-well scintillation counter</td></tr><tr><td>HiTrap SP column</td><td>GE Healthcare</td><td>17115101</td><td></td></tr><tr><td>Sertraline</td><td>Sigma</td><td>S6319</td><td></td></tr></tbody></table>

thermostabilization, expression, purification, and crystallization of the human serotonin transporter bound to s-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

A library of single point mutants in the SERTTC background was created to screen for thermostabilizing mutations. Individual mutants were generated using standard mutagenesis. The screening protocol utilizes transiently transfected HEK293S cells and a scintillation proximity-based thermostability screen to rapidly identity useful mutations for crystallization as outlined in Figure 1A. Plotting Tm values versus bound [3H]citalopram at RT reveals constructs with high thermostability and expression levels suitable for protein purification (Figure 1B). Three mutants (Y110A, I291A, and T439S) were combined to generate a highly stable construct (Figure 1C). Thermostability is also correlated with increased stability in short chain detergents necessary for the crystallization of the SERT-Fab complex.
The large scale expression of human SERT using baculovirus-transduced HEK293S GnTI- cells can take less than 2 weeks and can produce milligram quantities, as illustrated in Figure 2A. Use of the GFP-tagged SERTCC protein allows SERT to be conveniently followed during expression and purification by fluorescence (Figure 2B). Our purification strategy involved 1) solubilization of SERT bound to S-citalopram from HEK293S GnTI- cells in C12M in the presence of CHS as a stabilizing lipid; 2) binding of SERT to a Strep affinity matrix; 3) removal of contaminant proteins by extensive washing; and 4) elution of the functional SERT with buffer containing desthiobiotin (Figure 2C). The eluted protein is largely free of other detectable proteins by Coomassie blue staining and monodisperse as judged by FSEC (Figure 2D,&#160;E).
A similar strategy was taken to purify SERT with a Strep II tag which was used for reconstitution and immunization (Figure 3A,&#160;B). Incorporation of SERT into proteoliposomes boosts the serum half-life and stability of SERT and improves the likelihood of isolation of high-affinity antibodies. Furthermore, inclusion of lipid A, a component of the bacterial cell wall, serves as a potent adjuvant9. Multilamellar liposomes were prepared by the addition of buffer to a dried lipid mixture in glass tubes and resuspended in buffer. Extrusion of the liposomes through 200 nm pore size filters produces monodisperse unilamellar liposomal suspensions. The liposomes are then saturated with detergent followed by the addition of purified SERT in detergent. Finally, detergent is removed by addition of hydrophobic absorption resin to the lipid:detergent mixture. Additional ligand should be added to the reconstituted sample to select for antibodies that recognize the antidepressant bound conformation. The presence of SERT in the proteoliposomes should be confirmed by solubilizing a small sample with SDS-PAGE loading dye or C12M and running on SDS-PAGE and FSEC (Figure 3C,&#160;D).
Hybridoma cell lines expressing SERT&#160;antibodies can be screened for high-affinity binders which recognize 3D epitopes. These properties are crucial to the eventual success of crystallization, as the antibody must remain firmly bound to a structured region to promote crystal packing of homogeneous, well-ordered domains. In the first step, antibodies which recognize unstructured regions are identified. SERT is denatured and blotted onto a nitrocellulose membrane; antibodies which bind denatured SERT will be western-positive and likely recognize linear epitopes. In Figure 4A, we show 2 examples of antibodies which are western-positive and likely not useful to promote crystallogenesis. In Figure 4B, the remaining western-negative antibodies are incubated with 100 nM SERT-GFP and separated by FSEC. Antibodies which bind SERT will shift the GFP-positive peak to an earlier position. The SERT-antibody complexes may be diluted further in detergent to determine if they can bind with nanomolar affinity followed by analysis by FSEC. Addition of serotonin results in conformational changes in the transporter and thus the antibodies can be rescreened to determine if they can specifically recognize the SSRI-bound conformation. In Figure 4C, the antibodies are shown to bind SERT in the presence of serotonin, showing that the epitope(s) do not change from the SSRI to substrate bound state. Finally, in Figure 4D combinations of antibodies are tested for their ability to bind distinct epitopes, resulting in a further leftward shift. Here the 15B8 or 8A11 antibodies recognize an epitope which is different from 8B6.
The 8B6 antibody was chosen for further structural analysis based on preliminary crystal screening with papain treated Fab. The genes of the 8B6 Fab were cloned into an insect cell expression vector. Fab can be expressed and secreted from Sf9 cells growing in suspension. The 8B6 Fab can be purified from Sf9 cell supernatant by His-tag affinity (Figure 5A,&#160;B) and cation exchange chromatography (Figure 5C,&#160;D) resulting in protein which appears free of contaminants on SDS-PAGE gels. In Figure 5E, the recombinant 8B6 Fab is shown to bind SERT and is used in subsequent biochemical and biophysical experiments.
The affinity purified SERTCC is digested with thrombin and EndoH and mixed with 8B6 Fab to form a complex in the presence of S-citalopram. The transporter-antibody complex is then separated by SEC in C8M (Figure 6A) and the peak fractions contain both SERT and Fab as shown by SDS-PAGE (Figure 6B). Use of C8M is crucial for crystal formation probably because the short chain detergent allows better packing between molecules in the crystal lattice. FSEC is employed to determine which fractions should be pooled for crystallization (Figure 6C); fractions which are not monodisperse and/or contain large amounts of free SERT or Fab should not be combined.
Prism shaped SERT-antibody crystals can be grown in the presence of S-citalopram using this protocol by hanging drop vapor diffusion (Figure 7A). The resulting crystals diffract X-rays to a resolution of 3.15 &#197;10 (Figure 7B).
<img alt="Figure 1" src="/files/ftp_upload/54792/54792fig1.jpg" /> 
Figure 1: Scintillation Proximity-based Thermostability Assay. A. Overview of protocol for screening thermostability in the presence of [3H]citalopram. B. Maximum bound [3H]citalopram versus apparent melting temperature (Tm). Dotted lines represent values for the WT transporter. The 3 most thermostable mutants are labeled. Grey area represents mutants that have less than 10% of [3H]citalopram binding relative to WT and thus inaccurate Tm values due to a low signal-to-noise. C. Thermostability curves for WT SERTTC and the top 3 mutants. Error bars represent standard deviation (S.D.). <a href="http://cloudflare.jove.com/files/ftp_upload/54792/54792fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54792/54792fig2.jpg" /> 
Figure 2: Overview of Mammalian Heterologous Protein Expression. A. Schematized overview of BacMam virus generation and expression of SERT in HEK293S GnTI- cells. B. HEK293S GnTI- cells expressing the SERTCC (GFP fluorescence). C. Elution profile of SERTCC on Strep affinity resin. Green trace represents the concentration of desthiobiotin, 0 - 100% (0 - 5 mM). D. Analysis of affinity purified SERTCC on a 4 - 15% SDS-PAGE gel. E. FSEC of affinity purified SERTCC detected by GFP fluorescence (Excitation: 480 nm; Emission: 510 nm). The peak eluting at 15 mL is SERT (#) and 18 mL is free GFP (*). <a href="http://cloudflare.jove.com/files/ftp_upload/54792/54792fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54792/54792fig3.jpg" /> 
Figure 3: Representative Affinity Purification and Reconstitution of the SERTIC. A. Schematic overview of antibody generation. B. Elution profile observed at 280 nm of the affinity purification of SERTIC on Strep affinity resin. Green trace represents the concentration of desthiobiotin, 0 - 100% (0 - 5 mM). C. Analysis of affinity purified and reconstituted SERT on a 4 - 15% SDS-PAGE gel. D. FSEC of solubilized SERT following reconstitution. The fluorescence of tryptophan residues was used to detect SERT (Excitation: 280 nm; Emission: 335 nm). <a href="http://cloudflare.jove.com/files/ftp_upload/54792/54792fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54792/54792fig4.jpg" /> 
Figure 4: Analysis of Representative SERT Antibodies. A. Screening of antibodies by western blot. Approximately 1 &#181;g of SERTCC with or without GFP was applied to a 4 - 15% SDS-PAGE gel and blotted onto a nitrocellulose membrane. Binding was detected using a goat anti-mouse antibody conjugated to IR Dye. 2G4 and 10F2 are western positive. B. Binding of antibodies to 100 nM GFP-tagged SERT and detection by FSEC detected using GFP fluorescence. C. Binding of selected Fabs to 100 nM GFP-tagged SERT in the presence of 1 mM serotonin. D. Binding of 8A11 or 15B8 Fabs to SERT-8B6 Fab. Minor peaks eluting at 18 mL are free GFP. <a href="http://cloudflare.jove.com/files/ftp_upload/54792/54792fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54792/54792fig5.jpg" /> 
Figure 5: Representative Purification of the 8B6 Fab from Sf9 Cells. A. Elution profile observed at 280 nm of the purification of the 8B6 Fab by His-tag affinity chromatography. Green trace represents the concentration of imidazole, 0 - 50% (0 - 250 mM). B. Non-reducing and reducing SDS-PAGE gel after His-tag affinity purification. Protein which runs near 50 kDa is non-reduced Fab (#) and minor species at 25 kDa is reduced Fab (*). C. Elution profile observed at 280 nm of the purification of the 8B6 Fab by cation exchange displaying a single symmetrical peak which elutes under a linear sodium chloride gradient. Green trace represents the concentration of NaCl, 0 - 100% (0 - 500 mM). D. Analysis of the 8B6 Fab on a 12.5% SDS-PAGE gel following purification by cation exchange. E. Binding of the 8B6 Fab to 10 nM GFP-tagged SERT, detected using fluorescence of GFP. <a href="http://cloudflare.jove.com/files/ftp_upload/54792/54792fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54792/54792fig6.jpg" /> 
Figure 6: Representative Gel Filtration Chromatography of the SERT-8B6 Complex in the Presence of S-citalopram. A. Gel filtration elution profile of purified SERT-8B6 complex. Main peak eluting at 11.5 mL is the SERT-8B6 complex. Peak at 15 - 17 mL contains GFP and Fab. B. Analysis of the purified SERT-8B6 complex on a 4 - 15% SDS-PAGE gel. The positions of SERT and the heavy and light chains of the Fab are shown by a dash. C. FSEC of the size-separated fractions. SERT-8B6 complexes were detected using tryptophan fluorescence. Fraction 17 contains a larger amount of SERT which did not complex with Fab. <a href="http://cloudflare.jove.com/files/ftp_upload/54792/54792fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54792/54792fig7.jpg" /> 
Figure 7: Crystallization of the SERT-8B6 Complex&#160;Bound to S-citalopram. A. Light microscopy of parallelepiped shaped crystals of the SERT-8B6 complex after 2 weeks of growth. Scale bar equals 200 &#181;m. B. SERT-8B6 crystals diffract X-rays to 3.15 &#197;. Blue ring represents 3.15 &#197;. <a href="http://cloudflare.jove.com/files/ftp_upload/54792/54792fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: A Crystallization Screen for the SERT-8B6 Complex&#160;Bound to S-citalopram. <a href="http://cloudflare.jove.com/files/ftp_upload/54792/54792table1.xlsx">Please click here to download this table.</a>

Watch this Scientific Journal Video about Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram at JoVE.com

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

thermostabilization-expression-purification-crystallization-human

Research

JoVE Journal

Biochemistry

15.0K Views.  Oregon Health & Science University. This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

Video: Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

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 ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

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 ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Strategies for Expressing and Purifying Solute Carrier Transporters

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Immunology and Infection

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Biology

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Summary

Explore More Videos

PLAYLIST