Name: production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad
Uploaded: 2016-12-30T15:00:00
Description: Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

We thank the staff at the beamline X06DA at the Swiss Light Source, Paul-Scherrer-Institute, Villigen, Switzerland for support during synchrotron data collection. We are grateful to Monika Gompert for excellent technical support. The project was supported by the University of Cologne and grant INST 216/682-1 FUGG from the German Research Council. A PhD fellowship from the International Graduate School in Development Health and Disease to C.P. is acknowledged. The research leading to these results has received funding from the European Community&#39;s Seventh Framework Program (FP7/2007-2013) under grant agreement No. 283570 (BioStruct-X).

1. Cloning and Construct Design
<ol>
	<li>Clone the codon-optimized sequence (for E. coli) of C. difficile PPEP-1 without the signal peptide [amino acids 27-220, named hereafter recombinant PPEP-1 (rPPEP-1)11] into the pET28a vector using NdeI and XhoI restriction sites (Figure 1) with a stop codon at the 3&#39;-end (resulting vector pET28a-NHis-rPPEP-1). This produces a N-terminally 6xHis-tagged protein (NHis-rPPEP-1) with a thrombin cleavage site allowin...

<ol>
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G., Gardner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+and+Clostridium+difficile+infection:+a+review.">Mortality and Clostridium difficile infection: a review.</a> Antimicrob Resist Infect Control. 1 (1), (2012).</li><li>George, W. L., Sutter, V. L., Finegold, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+agent-induced+diarrhea--a+bacterial+disease.">Antimicrobial agent-induced diarrhea--a bacterial disease.</a> J Infect Dis. 136 (6), 822-828 (1977).</li><li>George, R. 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=Identification+of+Clostridium+difficile+as+a+cause+of+pseudomembranous+colitis.">Identification of Clostridium difficile as a cause of pseudomembranous colitis.</a> Br Med J. 1 (6114), 695 (1978).</li><li>Bartlett, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrative+review:+the+new+epidemic+of+Clostridium+difficile-associated+enteric+disease.">Narrative review: the new epidemic of Clostridium difficile-associated enteric disease.</a> Ann Intern Med. 145 (10), 758-764 (2006).</li><li>Kelly, C. P., LaMont, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile--more+difficult+than+ever.">Clostridium difficile--more difficult than ever.</a> N Engl J Med. 359 (18), 1932-1940 (2008).</li><li>&#220;nal, C. M., Steinert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+therapeutic+strategies+for+Clostridium+difficile+infections.">Novel therapeutic strategies for Clostridium difficile infections.</a> Expert Opin Ther Targets. 20 (3), 269-285 (2016).</li><li>Kelly, C. P., 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+Monoclonal+Antibody,+Bezlotoxumab+Targeting+C.+difficile+Toxin+B+Shows+Efficacy+in+Preventing+Recurrent+C.+difficile+Infection+(CDI)+in+Patients+at+High+Risk+of+Recurrence+or+of+CDI-Related+Adverse+Outcomes.">The Monoclonal Antibody, Bezlotoxumab Targeting C. difficile Toxin B Shows Efficacy in Preventing Recurrent C. difficile Infection (CDI) in Patients at High Risk of Recurrence or of CDI-Related Adverse Outcomes.</a> Gastroenterology. 150 (4), S122 (2016).</li><li>Tsutsumi, L. S., Owusu, Y. B., Hurdle, J. G., Sun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+discovery+of+treatments+for+C.+difficile+infection:+A+clinical+and+medicinal+chemistry+review.">Progress in the discovery of treatments for C. difficile infection: A clinical and medicinal chemistry review.</a> Curr Top Med Chem. 14 (1), 152-175 (2014).</li><li>Hensbergen, P. 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=Clostridium+difficile+secreted+Pro-Pro+endopeptidase+PPEP-1+(ZMP1/CD2830)+modulates+adhesion+through+cleavage+of+the+collagen+binding+protein+CD2831.">Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831.</a> FEBS Lett. 589 (24), 3952-3958 (2015).</li><li>Cafardi, V., 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=Identification+of+a+novel+zinc+metalloprotease+through+a+global+analysis+of+Clostridium+difficile+extracellular+proteins.">Identification of a novel zinc metalloprotease through a global analysis of Clostridium difficile extracellular proteins.</a> PLoS One. 8 (11), e81306 (2013).</li><li>Hensbergen, P. 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=A+novel+secreted+metalloprotease+(CD2830)+from+Clostridium+difficile+cleaves+specific+proline+sequences+in+LPXTG+cell+surface+proteins.">A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins.</a> Mol Cell Proteomics. 13 (5), 1231-1244 (2014).</li><li>Schacherl, M., Pichlo, C., Neundorf, I., Baumann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Basis+of+Proline-Proline+Peptide+Bond+Specificity+of+the+Metalloprotease+Zmp1+Implicated+in+Motility+of+Clostridium+difficile.">Structural Basis of Proline-Proline Peptide Bond Specificity of the Metalloprotease Zmp1 Implicated in Motility of Clostridium difficile.</a> Structure. 23 (9), 1632-1642 (2015).</li><li>Rawlings, N. D., Waller, M., Barrett, A. J., Bateman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEROPS:+the+database+of+proteolytic+enzymes,+their+substrates+and+inhibitors.">MEROPS: the database of proteolytic enzymes, their substrates and inhibitors.</a> Nucleic Acids Res. 42 (Release 10.0), D503-D509 (2014).</li><li>Bergfors, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeds+to+crystals.">Seeds to crystals.</a> J Struct Biol. 142 (1), 66-76 (2003).</li><li>Dauter, Z., Dauter, M., Dodson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jolly+SAD.">Jolly SAD.</a> Acta Crystallogr D Biol Crystallogr. 58 (Pt 3), 494-506 (2002).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Bradford, M. 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+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Crystallogr D Biol Crystallogr. 66 (Pt 2), 125-132 (2010).</li><li>Adams, P. 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=PHENIX:+a+comprehensive+Python-based+system+for+macromolecular+structure+solution.">PHENIX: a comprehensive Python-based system for macromolecular structure solution.</a> Acta Crystallogr D Biol Crystallogr. 66, 213-221 (2010).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallogr D Biol Crystallogr. 66 (Pt 4), 486-501 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL Molecular Graphics System. , (2002).</li><li>Pettersen, E. F., 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=UCSF+Chimera--a+visualization+system+for+exploratory+research+and+analysis.">UCSF Chimera--a visualization system for exploratory research and analysis.</a> J Comput Chem. 25 (13), 1605-1612 (2004).</li><li>Wang, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+phase+ambiguity+in+macromolecular+crystallography.">Resolution of phase ambiguity in macromolecular crystallography.</a> Methods Enzymol. 115, 90-112 (1985).</li><li>McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. 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C., Minor, W. <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+crystallography+over+the+past+decade+for+novel+drug+discovery+-+where+are+we+heading+next?.">X-ray crystallography over the past decade for novel drug discovery - where are we heading next?.</a> Expert Opin Drug Discov. 10 (9), 975-989 (2015).</li><li>Rubino, J. T., 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=Structural+characterization+of+zinc-bound+Zmp1,+a+zinc-dependent+metalloprotease+secreted+by+Clostridium+difficile.">Structural characterization of zinc-bound Zmp1, a zinc-dependent metalloprotease secreted by Clostridium difficile.</a> J Biol Inorg Chem. 21 (2), 185-196 (2016).</li><li>Carson, M., Johnson, D. H., McDonald, H., Brouillette, C., Delucas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=His-tag+impact+on+structure.">His-tag impact on structure.</a> Acta Crystallogr D Biol Crystallogr. 63 (Pt 3), 295-301 (2007).</li><li>Gasteiger, E., Walker, J. 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=Protein+Identification+and+Analysis+Tools+on+the+ExPASy+Server.">Protein Identification and Analysis Tools on the ExPASy Server.</a> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Dummler, A., Lawrence, A. 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X-ray crystallography is still the fastest and most accurate method to determine three-dimensional near-atomic resolution structures of proteins28. However, it requires the growth of well-ordered single crystals. These are often difficult to get and the crystalline state is artificial. However, a comparison of protein structures determined by X-ray crystallography with those determined by other methods, especially NMR, shows generally a very good agreement. In the case of PPEP-1, an NMR structure published rec...

Clostridium difficile is one of the major causes of nosocomial antibiotic-associated diarrhea infections1. This Gram-positive anaerobic bacterium is transmitted through its spore form via the fecal-oral route. In the past decade, new &#39;&#39;epidemic&#39;&#39; or &#39;&#39;hypervirulent&#39;&#39; strains (e.g. BI/NAP1/027) caused a drastic increase in new infections and fatality rates in North America and Europe2. C. difficile-associated disease (CDAD) is a life threatening colon inflammation with high fatality rates3. The symptoms range from diarrhea4 to pseudomembranous colitis5 and the often-fatal toxic megacolon6.
Treatment of CDAD is difficult as the virulent strains are multidrug-resistant and the recurrence rate is high7. At the moment therapy includes the antibiotics metronidazole, fidaxomicin or vancomycin, or in repetitively recurrent cases fecal microbiota transplantation. New therapeutic strategies are urgently needed8. Some progress is recorded as the therapeutic monoclonal antibody Bezlotoxumab, targeting C. difficile toxin B9, has recently successfully passed phase III clinical trials and was filed for approval with the FDA and EMA. Additionally, new antibiotics are being tested at the moment at different stages of clinical trials10.
To develop effective treatment new therapeutic targets must be identified. The recently discovered C. difficile protease proline-proline endopeptidase-1 (PPEP-1; CD2830/Zmp1; E.C. 3.4.24.89) is such a promising target, as the lack of PPEP-1 in a knock-out strain decreases virulence of C. difficile in vivo11. PPEP-1 is a secreted metalloprotease12,13 cleaving two C. difficile adhesins at their C-terminus13 thus releasing the adherent bacteria from the human gut epithelium. Therefore, it is involved in maintaining the balance between the sessile and motile phenotype of C. difficile. To develop selective inhibitors against PPEP-1 and to understand how it recognizes its substrates intimate knowledge of its three-dimensional structure is indispensable. We have solved the first crystal structure of PPEP-1 alone and in complex with a substrate peptide14. PPEP-1 is the first known protease that selectively cleaves peptide bonds between two proline residues15. It binds the substrate in a double-kinked manner and stabilizes it via an extended aliphatic-aromatic network of residues located in the S-loop that covers the protease active site14. This substrate-binding mode is unique to PPEP-1 and not found in human proteases to date. This makes it a promising drug target, and off-target effects of inhibitors very unlikely.
To develop and screen selective PPEP-1 inhibitors in the future a large amount of pure and monodisperse PPEP-1 protein is needed. Furthermore, to determine the mode of binding of first inhibitors, co-crystal structures with PPEP-1 will have to be determined. In our hands PPEP-1 constantly produces intergrown crystals. Thus we have developed an optimization procedure to produce single diffraction-quality crystals of PPEP-1. In this protocol we describe in detail the production, purification, crystallization and structure solution of PPEP-114. We use intracellular expression in Escherichia coli of a PPEP-1 variant lacking the secretion signal sequence, affinity chromatography and size exclusion chromatography with removal of the purification tag, followed by microseeding16 into an optimization screen and structure determination via zinc single-wavelength anomalous dispersion (zinc-SAD)17. This protocol can be adapted for production and structure determination of other proteins (e.g. metalloproteases) and in particular for proteins producing intergrown crystals. On request, plasmid DNA of the construct (pET28a-NHis-rPPEP-1) and diffraction data can be provided for educational purposes.

<table border="0" cellpadding="0" cellspacing="0" width="657">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Genes / Vectors / cell strains</td>
			<td style="width:83px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET28a vector</td>
			<td>Merck-Millipore</td>
			<td>69864</td>
			<td>Thrombin cleavable N-terminal His-tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E. coli strain BL21 (DE3) Star</td>
			<td>ThermoFisher Scientific</td>
			<td>C601003</td>
			<td>RNase H deficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Codon-optimized gene (for E. coli) of PPEP-1 (CD630_28300)</td>
			<td>Geneart (Thermo Fisher Scientific)</td>
			<td>custom</td>
			<td>amino acids 27-220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast extract</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antifoam B</td>
			<td>Sigma-Aldrich</td>
			<td>A5757</td>
			<td>aqueous-silicone emulsion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>AppliChem</td>
			<td>A1008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1087</td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>any</td>
			<td></td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNaseI</td>
			<td>AppliChem</td>
			<td>A3778</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>AppliChem</td>
			<td>A1073</td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>215996</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol 100%</td>
			<td>any</td>
			<td></td>
			<td>purest grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td>any</td>
			<td></td>
			<td>for storage and cryocooling of crystals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment (general)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking incubator</td>
			<td>any</td>
			<td></td>
			<td>providing temperatures of 20 &#176;C - 37 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glassware</td>
			<td>any</td>
			<td></td>
			<td>baffled Erlenmeyer flasks (50 ml - 2.8L)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge for large culture volumes</td>
			<td>any</td>
			<td></td>
			<td>centrifuge for processing volumes up to 12 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator Vibra-Cell VCX500</td>
			<td>Sonics</td>
			<td>SO-VCX500</td>
			<td>or any other sonicator / cell disruptor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultracentrifuge</td>
			<td>any</td>
			<td></td>
			<td>centrifuge providing speeds up to 150.000 x g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NiNTA Superflow resin</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Empty Glass Econo-Column</td>
			<td>Bio-Rad</td>
			<td>7371007</td>
			<td>or any other empty glass or plastic column</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Size exclusion chromatography column HiLoad Superdex 200 16/600</td>
			<td>GE Healthcare</td>
			<td style="width:136px;">28989335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromatography system &#196;kta Purifier</td>
			<td>GE Healthcare</td>
			<td>28406264</td>
			<td>or any other chromatography system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dialysis tubing Spectra/Por 3</td>
			<td>Spectrum Labs</td>
			<td>132724</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dialysis tubing closures</td>
			<td>Spectrum Labs</td>
			<td>132738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrafiltration units (concentrators) 10.000 NWCO</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV-Vis spectrophotometer</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment (crystallography)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low volume pipette 0.1-10 &#181;l</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Positive displacement pipette Microman M10</td>
			<td>Gilson</td>
			<td>F148501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Crystallization robot</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well crystallization plates TTP IQ with three protein wells</td>
			<td>TTP</td>
			<td style="width:136px;">4150-05810</td>
			<td>or any other 96-well crystallization plate&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well CombiClover Junior Plate</td>
			<td>Jena Bioscience</td>
			<td>EB-CJR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Crystal Clear Sealing Tape</td>
			<td>Hampton Research</td>
			<td>HR3-511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Siliconized Glass Cover Slides</td>
			<td>Hampton Research</td>
			<td>HR3-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal</td>
			<td>Hampton Research</td>
			<td>diverse</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial crystallization screens: Wizard, PACT++, JCSG++</td>
			<td>Jena Bioscience</td>
			<td>diverse</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JBS Beads-for-Seeds</td>
			<td>Jena Bioscience</td>
			<td>CO-501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalCap SPINE HT (nylon loops)</td>
			<td>Hampton Research</td>
			<td>diverse</td>
			<td>loop sizes 0.025 mm - 0.5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalCap Vial</td>
			<td>Hampton Research</td>
			<td>HR4-904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cryogenic Foam Dewar 800 ml</td>
			<td>Hampton Research</td>
			<td>HR4-673</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cryogenic Foam Dewar 2L</td>
			<td>Hampton Research</td>
			<td>HR4-675</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vial Clamp, Straight</td>
			<td>Hampton Research</td>
			<td>HR4-670</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalWand Magnetic, Straight</td>
			<td>Hampton Research</td>
			<td>HR4-729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoCane 6 Vial Holder</td>
			<td>Hampton Research</td>
			<td>HR4-711</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoSleeve</td>
			<td>Hampton Research</td>
			<td>HR4-708</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoCane Color Coder - White</td>
			<td>Hampton Research</td>
			<td>HR4-713</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Straight microforcep</td>
			<td>any</td>
			<td></td>
			<td>for manipulation of sealing tape. etc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acupuncture needle</td>
			<td>any</td>
			<td></td>
			<td>e.g. from a pharmacy</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Stereo microscope</td>
			<td>any</td>
			<td></td>
			<td style="width:191px;">for inspection of crystallization plates and crystal mounting, magnification up to 160X</td>
		</tr>
	</tbody>
</table>

production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

rPPEP-1 is overexpressed in several E. coli strains, with the highest yield in E. coli BL21 (DE3) Star (Figure 1C). After the first NiNTA affinity chromatography step the 6xHis-tag can be successfully cleaved off from most of the protein and in the second NiNTA step undigested protein can be completely separated from thrombin-digested protein (Figure 1D). On a S200 16/600 column untagged rPPEP-1 migrates as monomer with occasional fronti...

Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

The overall goal of this procedure is to efficiently grow single lattice diffraction quality crystals of recombinant proline proline endopeptidase-1, or rPPEP-1, protein. Without this method, rPPEP-1 instantly produces highly intergrown crystals not suitable for xray diffraction analysis. The main advantage of this technique is that a high number of well ordered and well diffracting crystals for structural determination of rPPEP-1 can be produced in a short period of time and with limited material input.This procedure makes use of the microcity method and can be adapted for every successful initial crystallization condition of rPPEP-1 as well as its substrate peptide complexes. This method can be adapted to produce well ordered crystals of virtually every protein that yields intergrown crystals. It can also be used in cross seeding experiments, protein variance, or in cocrystallization experiments with small molecules.Visual demonstration of the handling of crystallization methods is crucial, since even small variations can have a significant impact on the diffraction quality of the crystals. Carry out crystallization trials in the sitting drop format using standard commercially available screens and a crystallization robot. First, concentrate the purified protein to 12 milligrams per milliliter using a centrifugal ultrafiltration device in five minute intervals at 4000 times g and four degrees Celsius.Mix the protein after each interval to prevent precipitation and aggravation. Determine the protein concentration at 280 nanometers by using the extinction coefficient of 25, 900 per molar centimeter. After equilibrating the protein to 20 degrees celsius clear away all particles and dust by centrifugation for 10 minutes at 16000 times g and 20 degrees celsius.To perform crystal screens, use prefilled crystallization plates sealed and stored at four degrees celsius. When setting up trials, work swiftly, as the small volumes quickly dry out. Also use a humidity chamber around the dock of the robot if possible.After equilibrating all crystallization plates to 20 degrees celsius, set up the screen by pipetting protein ent reservoir into subwells two to four. Use a drop volume of 300 nanoliters. Use protein to reservoir ratios of 200:100 for subwell two, 150:150 for subwell three, and 100:200 for subwell four.Immediately seal the plate and place it in a chamber at 20 degrees celsius. Inspect the trays after setup every day during the first week and then follow with weekly inspection. For a cocrystallization of the substrate peptide rPPEP-1 complexes, mix rPPEP-1 at 24 milligrams per milliliter in a 1:1 ratio with a sevenfold molar excess of peptide solution lay off lyophilized powder solubilized in tris buffered saline.After incubating for 30 minutes at 20 degrees celsius, clear away all particles and dust by centrifugation for 10 minutes at 16000 times g and 20 degrees celsius. Proceed with crystallization using the same microseeding procedure as for the unbound rPPEP-1 protein. Highly intergrown crystals of rPPEP-1 appeared after two days in a condition containing 2.4 molar ammonium phosphate dibasic and 0.1 molar trisapseal pH 8.5.To optimize the initial condition, use software to calculate the volumes and the pipetting scheme to obtain two milliliters of each condition that allows for 10 optimization screens. Conditions include 1.8 to 2.55 molar ammonium phosphate dibasic in steps of 0.15 molar as well as 0.1 molar trisapseal pH 7.5 to 9.0 in steps of 0.5 pH units. Prepare a grid screen comprising 24 conditions from stock solutions.Use the pipetting scheme to compose the individual conditions. Pipette 200 microliters of every grid screen solution into the wells of a 24 well plate and equilibrate the plate at 20 degrees celsius. Manually set up the crystallization plate.Use a drop volume of three microliters and a protein to reservoir ratio of 2:1, 1.5:1:5, and 1:2. Here, use a positive displacement pipette to avoid air bubble formation. Immediately seal the plate.Then place the plate in a chamber at 20 degrees celsius. After one to four days, highly intergrown crystals of rPPEP-1 appear in the four conditions containing 2.55 molar ammonium phosphate dibasic and 0.1 molar trisapseal pH 7.5 to 9.0, whereas no crystals are formed in the remaining 20 conditions. Perform the microseeding procedure to obtain single crystals of rPPEP-1 in these conditions.To prepare a microseed stock, first choose a single intergrown crystal from one of the successful conditions. Next, transfer 50 microliters of the respective mother liquor into a 1.5 milliliter tube containing a small, highly polished glass bead and one microliter of the mother liquor to the glass cover slide. Using a mounted nylon lube, fish out the crystal and transfer it into the drop on the glass cover slide.Then transfer the liquid containing the crystal into the tube and vortex at high speed for 30 seconds, making sure the glass bead is swirling. Make a 1:1000 dilution of the seed stock into a new 1.5 milliliter tube containing the same freshly prepared condition and vortex thoroughly for five seconds. Next, remove the seal of the plate covering the 20 conditions with clear drops.Pipette 0.5 microliters of the seed stock into the wells. Seal the plate and place it into a chamber at 20 degrees celsius. After applying this microseeding technique, single crystals of high diffraction quality appear several hours to several days after setup in various conditions, containing 1.8 to 2.4 molar ammonium phosphate and 0.1 molar tris pH 7.5 to nine.Crystals grow to a size of 100 to 200 microns in the largest dimension. Choose the optimal size of nylon loop for the maximal length of the chosen crystals by placing the loop on the sealing tape just above the crystal and focusing up and down. The typical longest access of rPPEP-1 crystals is about 100 to 200 microns.Also prepare the appropriate cryocondition. Fill the foam dewers with liquid nitrogen. Then load the vial clamp with a vial and precool it in the liquid nitrogen filled 800 milliliter foam dewer.Place a cryocane holder marked with a suitable identifier and a cryosleeve in the liquid nitrogen filled two liter foam dewer. Then load the magnetic wand with the mounted nylon loop of the right size. Next, cut open the sealing tape on the crystallization plate with a sharp scalpel and remove it with forceps.Pipette one microliter of the cryocondition onto the cover slide. It is particularly important to work fast at this step, as drying out the crystal or the tiny volume of cryosolution in the loop could lead to fluctuations in crystal quality or even a complete loss of diffraction. All crystal manipulation steps should be performed under the stereo microscope.If the crystal sticks to the plastic surface, detach it from the ground by deforming the surrounding plastic with an acupuncture needle. Remove the crystal from the drop by fishing it out with the mounted nylon loop. Quickly transfer the crystal to the drop of cryocondition and let it equilibrate for one second.Then fish the crystal out of the drop with the mounted nylon loop as quickly as possible. Immediately plunge freeze the retrieved crystal in liquid nitrogen. When the liquid nitrogen around the mounted loop stops boiling, place the loop in the vial.Place the vial on the cryocane holder. Once the holder is loaded with six vials, place a cryosleeve around the holder. Store the crystals in a tank filled with liquid nitrogen until use.To perform data collection, carefully retrieve the mounted crystal from the cryocane using a clamp and store it in a foam dewer for transport. Move the beam stop and the detector into the park position and move the cryonozzle up by some millimeters. Mount the base of the cryocap onto the goniometer head by retaining the base with one's fingers while quickly removing the vial.Then move the cryonozzle and beam stop back into place and center the crystal. At all times, be careful not to touch the beam stop or detector. Proceed to collect the 180 degree data set with an oscillation angle of 0.1 degrees, as shown here.Shown here are highly intergrown rPPEP-1 crystals obtained from initial screening using commercial crystallization screens in 1.4 sodium citrate tribasic dihydrate, 0.1 molar HEPES sodium, pH 7.5. Shown here are crystals obtained from initial screening with 60%volume to volume tacsimate, pH 7.0, 0.1 molar BIS-TRIS propane, pH 7.0. Crystals also appeared in 2.4 molar ammonium phosphate dibasic, 0.1 molar tris, pH 8.5.After applying the microseeding optimization procedure, single crystals were contained in several conditions, including 2.1 molar ammonium phosphate dibasic, 0.1 molar tris, pH 8, and 2.25 molar ammonium phosphate dibasic, 0.1 molar tris, pH 8. The crystals mounted in nylon loops are of 100 to 200 microns in size and appear to have a single lattice from microscopic inspection. Xray diffraction analysis shows that these crystals indeed exhibit a single lattice and diffract xrays to near atomic resolution.Solving the crystal structure of the recombinant PPEP-1 substrate peptide complex gives insight into the substrate binding mode of PPEP-1. The substrate peptide could diffuse into the substrate binding group of PPEP-1 in its open confirmation. Then as loop closure would occur, the substrate interacts with PPEP-1 via hydrogen bonds and the unique aliphatic-aromatic side chain network located on the S-loop.The substrate binds in a unique double kinked conformation with kinks at the sizzle peptide bond and the P2 prime to P3 prime peptide bond. After watching this video, you should have a good understanding of how to produce single lattice well diffracting crystals of recombinant PPEP-1 for structural studies. Similar approaches can be used with other proteins, yielding highly intergrown crystals and further varied incubation temperature and C-stock dilution.Due to its versatility and simple use, this simple technique should be tried in every crystal optimization process.

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