Shiga+Toxin+subunit+B+(Shigella+dysenteriae)

*NCBI Gene # or RefSeq#: 3796556 *Protein ID (NP or XP #) or Wolbachia#: NC_007606.1 *Organism (including strain): S. dysenteriae Etiologic Risk Group (see link below): Risk Group 2 Bacterial Agents including Chlamydia [|__https://my.absa.org/tiki-index.php?page=Riskgroups__]
 * Target (protein/gene name): Shiga Toxin Subunit B

*Disease Information (sort of like the Intro to your Mini Research Write up): Shigellosis is an infectious foodborne illness caused by bacteria of the Escherichia and Shigella genus. This infectious disease is usually transmitted by physical contact between humans and a hand to mouth contact for the bacteria to infect humans. Food is another source of transmission. Symptoms of Shigella may vary from abdominal discomfort to cramps, diarrhea, slimy stools, fever, pus or mucus in stools, tenesmus, or seizures. Shigella is a deadly disease that prevalently affects children of developing countries. Furthermore, Shigella is resistant to traditional first line antibiotics such as ampicillin.

Link to TDR Targets page (if present): n/a

Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) [|__http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=622__]

Essentiality of this protein: The enzyme induces a cytotoxic enzyme that inhibits protein synthesis through inactivating the ribosomes within target cells. The enzyme targets the cell surface of the target cell and undergoes endocytosis and transport to the endoplasmic reticulum. Then the active part is set free and translocated to the cytosol dependent on several factors. In the cytosol the toxin attacks its target.

Is it a monomer or multimer as biological unit? (make prediction at [] ) Most probable biological assembly: pentamer []

Complex of proteins?:  Yes Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Pteroic acid (PTA) and 4-[3-(2-amino-1,4-dihydro-6-hydroxy-4-oxo-5-pyrimidinyl)propyl]-benzoic acid (PBA) has been found to be a drug candidate that have yielded low IC50 values in enzyme assays. [|__http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929769/__] *EC#: 3.2.2.22 Link to BRENDA EC# page: [|__http://www.brenda-enzymes.org/enzyme.php?ecno=3.2.2.22__] Figure 1. Reaction catalyzed by rRNA N-glycosylase (3.2.2.22). Screenshot from BRENDA.

Enzyme Assay information (spectrophotometric, coupled assay, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) [|__http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929769/__] Ricin assay is performed by using Shiga toxin to inhibit translational ribosomal proteins from translating luciferase. -- links to assay reagents (substrates) pages. No reagents involved in this assay. Ricin assay was used instead. Link about assay included above. --- List cost and quantity of substrate reagents, supplier, and catalog # <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Cost and Quantity: $275.50 for 0.5mg <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Supplier: Sigma Aldrich <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Catalog #: SML0562

Structure (PDB or Homology model) [|__http://www.rcsb.org/pdb/explore/explore.do?structureId=1DM0__] <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Figure 2. PDB Structure of Shiga Toxin (PDBID: 1DM0).

-- PDB # or closest PDB entry if using homology model <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">PDBID: 1DM0

<span style="background-color: #ffffff; font-family: Arial; font-size: 13.3333px; vertical-align: baseline;">Current Inhibitors: List of Inhibitors [|__http://www.brenda-enzymes.org/enzyme.php?ecno=3.2.2.22#INHIBITORS__]
 * 1) ((5-(2-amino-4,6-dihydroxy-5-pyrimidinyl)pentanoyl)amino)acetic acid
 * 2) 2-methylsulfonyl-1-(naphthalen-1-ylmethyl)benzimidazole
 * 2,2-dimethyl-4-[(E)-2-phenylethenyl]-2,3-dihydro-1H-1,5-benzodiazepine

Expression Information (has it been expressed in bacterial cells): <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">This protein has been expressed in E. coli and S. dysenteriae cells. Purification Method: <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">The supernatant was filter sterilized with a 0.22 µm syringe filter before being applied to the chitin column for purification. [|__http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929769/__]

Image of protein (PyMol with features delineated and shown separately): <span style="background-color: #ffffff; color: #ff9900; font-family: Arial; font-size: 12px; vertical-align: baseline;"> <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Figure 3. Pymol representation of Shiga Toxin Subunits (PDBID: 1DM0) colored by chains shown in cartoon (1DM0.pdb).

*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">TPDCVTGKVE YTKYNDDDTF TVKVGDKELF TNRWNLQSLL LSAQITGMTV TIKTNACHNG GGFSEVIFR

*Length of your protein in Amino Acids: <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 13.3333px; vertical-align: baseline;">69 Molecular Weight of your protein in kiloDaltons using the [|__Expasy ProtParam__] website: 7690.66 Molar Extinction coefficient of your protein at 280 nm wavelength: <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Ext. coefficient 8605 <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Abs 0.1% (=1 g/l) 1.119, assuming all pairs of Cys residues form cystines

<span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Ext. coefficient 8480 <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Abs 0.1% (=1 g/l) 1.103, assuming all Cys residues are reduced

TMpred graph Image ( [|__http://www.ch.embnet.org/software/TMPRED_form.html__] ). <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;">Figure 4. TMpred graph of Shiga Toxin Subunit B.

*CDS Gene Sequence (paste as text only): <span style="background-color: #ffffff; color: #4a86e8; font-family: Verdana; font-size: 13.3333px; vertical-align: baseline;">>gi|82775382:1284822-1285091 Shigella dysenteriae Sd197 chromosome, complete genome <span style="background-color: #ffffff; color: #4a86e8; font-family: Verdana; font-size: 13.3333px; vertical-align: baseline;">ATGAAAAAAACATTATTAATAGCTGCATCGCTTTCATTTTTTTCAGCAAGTGCGCTGGCGACGCCTGATT <span style="background-color: #ffffff; color: #4a86e8; font-family: Verdana; font-size: 13.3333px; vertical-align: baseline;">GTGTAACTGGAAAGGTGGAGTATACAAAATATAATGATGACGATACCTTTACAGTTAAAGTGGGTGATAA <span style="background-color: #ffffff; color: #4a86e8; font-family: Verdana; font-size: 13.3333px; vertical-align: baseline;">AGAATTATTTACCAACAGATGGAATCTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATGACTGTA <span style="background-color: #ffffff; color: #4a86e8; font-family: Verdana; font-size: 13.3333px; vertical-align: baseline;">ACCATTAAAACTAATGCCTGTCATAATGGAGGGGGATTCAGCGAAGTTATTTTTCGTTGA

*GC% Content for gene: <span style="background-color: #ffffff; color: #4a86e8; font-family: Arial; font-size: 12px; vertical-align: baseline;"> 11.594203% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):

Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): ( <span style="background-color: #ffffff; color: #008000; font-family: Arial; font-size: 12px; vertical-align: baseline;">link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to. Primer design results for 'tail' primers (this is just 2 sequences): **