Target+- 5-nucleotidase+SurE+(Chlamydia+trachomatis)

**Target:** 5-nucleotidase SurE **RefSeq:** CT218 **Etiologic Risk Group**: Risk Group 2 **Background/Disease Information:** Trachoma is an infectious disease caused by Chlamydia trachomatis, and is theleading cause of infectious blindness in the world. Trachoma causes a roughening of the inner surface of the eyelids, eye discharge, eye scarring, ingrown eyelashes, and other painful symptoms. WHO Guidelines recommend the use of antibiotics such as Azithromycin or tetracycline to treat the infection. This affliction is currently classified as a neglected tropical disease [1]. **Essentiality of this protein:** "5-nucleotidases (members of EC 3.1.3.5 and EC 3.1.3.6) dephosphorylate non-cyclic nucleoside monophosphates to nucleosides and inorganic phosphate" [2] "DNA and RNA synthesis requires a continuous and balanced supply of the four deoxyribonucleotides and four ribonucleotides. A network of ... enzymes regulates the size of each pool with the enzymes ribonucleotide reductase, nucleoside kinases, and nucleotidases playing the main roles." [3]. By inhibiting the function of this enzyme, the regulation of nucleotides and phosphorus is depleted, leading to the organism's inability to continue essential cell functions. **Druggable** **Target**: There are several known inhibitors, indicating that it may be possible to drug this target using manufactured compounds [2]. **EC#:** 3.1.3.5 
 * Protein ID: **NP_219722.1
 * Organism **: //Chlamydia trachomatis// (D/UW-3/CX)
 * Complex of proteins: **No
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">Link to BRENDA EC# page: **<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;"> []

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Enzyme Assay information**: "Purified E. coli proteins were screened for phosphatase activity using general phosphatase substrate p-nitrophenyl phosphate (pNPP) in 96-well microplates. The reaction mixture (0.2 ml) contained 50 mM HEPES-K buffer (pH 7.5), 5 mM MgCl2, 0.5 mM MnCl2, 0.5 mM NiCl2, 40 mM pNPP, 0.1–1.0 g of purified protein. After addition of protein samples, the plates were incubated for 1–3 h at 37 °C before taking the reading at 410 nm" [2].

[|HEPES-K Salt] <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[|MgCl2] <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[|MnCl2] <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[|NiCl2] <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[|PPnPP] <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[|5nucleotidase SurE]
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">Link to Sigma page for assay or assay reagents (substrates): **

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Cost and Quantity of Substrate Reagents and Supplier:** <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">50mM HEPES-K Buffer (Sigma): $38.70/25g <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">5mM MgCl2 (Sigma): $43.60/100g <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">0.5mM MnCl2 (Sigma): $45.50/10g <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">0.5mM NiCl2 (Sigma): $36.00/50g <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">40mM pNPP (Sigma): $79.60/1g <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">0.1-1.0g <span class="wiki_link_ext">[|5nucleotidase SurE (Ontario Centre for Structural Proteomics):] No Price Given [2] <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">3'-CMP Cytidine-3'-monophosphate (Santa Cruz Biotechnology, Inc.): $98/1g

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Structure Available:** Homology Model <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Closest PDB Entry**: 1J9J <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;"> <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Query Coverage**: 65% <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Max % Identities**: 37% <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**% Positives:** 55% <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Chain Used for Homology:** Chain A
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">Homology Model Option: **

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Current Inhibitors:** pyrimidine nucleotide, nucleoside analogs, dTTP, dGTP, dCTP, dATP [2]. <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Expression Information**: Expressed in //E.coli// DH5α [2]. <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Purification Method**: "To prepare protein samples for screening, recombinant proteins containing an N-terminal His6 fusion tag were expressed in 1-liter E. coli BL21(DE3) cultures and affinity-purified on nickel-nitrilotriacetic acid resin (Qiagen) as previously described (17). Briefly, cell lysates were loaded onto small (1.5 x 4 cm) nickel columns equilibrated with loading buffer (50 mM HEPES-K, pH 7.5; 0.5 M NaCl; 5 mM imidazole), washed with 10 volumes of washing buffer (loading buffer containing 30 mM imidazole), and eluted with 250 mM imidazole in loading buffer" [2].

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">Image of Protein: **

<span style="font-family: 'Courier New',Courier,monospace;">MTETRRLRILITNDDGIKAKGISLLISLLREADFADLYVVAPLEEQSGRSMAFSLVEPTA <span style="font-family: 'Courier New',Courier,monospace;">LEPFDYPQRVQEAWAVTGTPVDCVKLAIGELFKENALDLILSGINNGKNSGRCLYYSATV <span style="font-family: 'Courier New',Courier,monospace;">GAIREANLHGIPAIALSQSENIAFFQKAHMASLIRSLCEFTVAYKHTDPLGLNVNFPAST <span style="font-family: 'Courier New',Courier,monospace;">DDSPWKGIRFTLSGNEFLFGIPRLVRTEGNRRYYTLYDMRDKVSEEFSEEYLALANNYIS <span style="font-family: 'Courier New',Courier,monospace;">AAPLVSKNTPRATLSEEELAFLKDSFEQSVLWKASLNLEEDLA
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">Amino Acid Sequence: **

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Length of protein in Amino Acids:** 283 <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Molecular Weight of protein in kiloDaltons:** 31544.8 kDa <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**Molar Extinction coefficient of your protein at 280 nm wavelength**: 31525 M-1 cm-1

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**TMpred graph Image:** <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%; line-height: 0px; overflow: hidden;">

<span style="font-family: 'Courier New',Courier,monospace;">ATGACAGAGACACGACGACTTCGCATTCTTATTACCAATGATGACGGTATCAAAGCCAAA GGAATCAGTCTGCTGATTTCTCTACTCCGTGAAGCTGACTTCGCCGATCTTTATGTAGTA GCCCCTTTAGAAGAACAGTCTGGGAGAAGTATGGCTTTCTCATTAGTAGAGCCAACTGCC CTTGAGCCTTTTGATTATCCTCAAAGAGTCCAAGAAGCTTGGGCTGTCACCGGCACTCCT GTTGACTGTGTGAAATTAGCTATAGGAGAACTCTTTAAAGAGAACGCTCTGGATCTTATT TTATCGGGTATTAATAATGGGAAAAATTCCGGACGCTGCCTTTATTACTCTGCCACTGTA GGAGCTATAAGAGAAGCGAATCTCCATGGAATTCCTGCGATAGCTCTTTCTCAAAGTGAG AATATCGCTTTTTTCCAAAAAGCTCATATGGCCTCCCTAATTCGTTCCTTATGCGAGTTT ACCGTTGCCTATAAGCACACCGATCCCTTAGGACTTAATGTAAATTTCCCTGCTAGCACG GACGATTCTCCATGGAAAGGAATTCGTTTCACACTTTCTGGAAATGAATTCTTATTCGGT ATCCCAAGATTAGTCCGTACGGAAGGAAATCGTCGTTACTATACGCTCTATGATATGCGA GATAAAGTATCCGAAGAGTTCTCTGAAGAGTATCTTGCTTTAGCAAATAACTATATTAGT GCGGCTCCACTAGTTTCTAAAAATACCCCTCGAGCAACCCTTTCCGAAGAAGAACTAGCT TTCCTAAAAGACTCTTTCGAACAATCTGTGTTGTGGAAAGCTTCTTTAAACTTAGAAGAA <span style="font-family: 'Courier New',Courier,monospace;">GATCTGGCTTAG
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">CDS Gene Sequence: **

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**GC% Content for gene**: 42%

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**CDS Gene Sequence:** <span style="font-family: 'Courier New',Courier,monospace;">ATGACCGAGA CTCGTCGACT TCGTATCCTA ATTACAAATG ATGATGGGAT TAAAGCTAAG GGCATTTCTC TTCTAATTTC CTTACTCCGC GAAGCGGATT <span style="font-family: 'Courier New',Courier,monospace;">TTGCTGATTT GTATGTGGTC GCCCCACTTG AGGAACAATC TGGACGATCT ATGGCTTTTT CTTTAGTAGA ACCTACAGCA CTAGAACCTT TCGACTATCC <span style="font-family: 'Courier New',Courier,monospace;">ACAAAGAGTA CAAGAGGCTT GGGCTGTTAC CGGAACACCT GTGGACTGTG TTAAATTAGC TATCGGGGAG TTATTTAAGG AAAATGCCCT CGACCTTATT <span style="font-family: 'Courier New',Courier,monospace;">CTTTCTGGTA TTAACAATGG AAAAAACTCA GGTCGTTGCT TGTATTATTC TGCCACCGTC GGAGCTATTC GTGAAGCAAA TCTTCACGGA ATCCCTGCAA <span style="font-family: 'Courier New',Courier,monospace;">TCGCACTTTC TCAGTCTGAG AACATCGCCT TTTTCCAGAA AGCCCACATG GCATCTTTGA TTCGATCTTT GTGCGAATTC ACCGTGGCTT ATAAACATAC <span style="font-family: 'Courier New',Courier,monospace;">TGACCCTTTA GGGTTAAATG TCAATTTTCC AGCTTCTACT GACGATTCTC CATGGAAAGG TATCCGCTTC ACATTATCCG GAAACGAATT CTTATTTGGT <span style="font-family: 'Courier New',Courier,monospace;">ATTCCTAGAT TAGTTCGCAC AGAAGGGAAC CGACGTTATT ACACCCTATA TGACATGCGT GATAAAGTCT CCGAGGAATT TTCAGAAGAA TATCTAGCAT <span style="font-family: 'Courier New',Courier,monospace;">TGGCTAATAA CTATATATCT GCTGCTCCTC TAGTATCAAA AAACACACCA AGAGCAACAC TTTCTGAGGA AGAATTGGCG TTCTTAAAAG ATTCCTTCGA <span style="font-family: 'Courier New',Courier,monospace;">GCAAAGCGTT CTTTGGAAGG CTTCTTTGAA TCTCGAAGAA GACCTCGCCT AA

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">**GC% Content for Gene:** 42.1%

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">NOT NECESSARY FOR SPRING **

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">Primer design results for 'tail' primers (this is just 2 sequences):

<span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[1] http://www.who.int/blindness/causes/trachoma/en/index.html <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[2] http://www.jbc.org/content/279/52/54687.full.pdf <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[3] http://www.jbc.org/content/278/47/46195.full.pdf <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">[4] http://patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=2754
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 120%;">References: **