TargetF15+-+Secreted+Phosphatase+(Francisella+tularensis)

Tularemia is an infectious disease caused by the bacteria Francisella tularensis, commonly found in animals such as rodents, rabbits and hares [4]. Humans can contract tularemia by means of getting bitten by an infected tick, deerfly, or other insect, handling an infected animal carcass, eating or drinking contaminated food or water, or breathing in the bacteria itself [4]. Tularemia ranges from mild to life-threatening, often with the accompanying symptoms of a sudden fever, chills, headaches, diarrhea, muscle aches, joint paint, dry cough, and progressive weakness [4]. More serious symptoms include development of pneumonia, chest pain, cough and difficulty breathing [4]. Tularemia occurs naturally in the United States and because of its highly infectious nature, it can be used as a bioterrorism weapon. [] Is it a monomer or multimer as biological unit ** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): 2-mer
 * *Target (protein/gene name): ** AcpA (secreted acid phosphatase)
 * *NCBI Gene # or RefSeq#: ** FTN_0090
 * *Protein ID (NP or XP #) or Wolbachia#: ** YP_897755.1
 * *Organism (including strain): ** //Francisella tularensis//
 * Etiologic Risk Group: ** Those who live or visit areas of Arkansas, Missouri, or Oklahoma due to the concentration of ticks. Also those who engage in hunting, trapping, gardening, landscaping, or work in wildlife management or veterinary medicine.
 * * Disease Information (sort of like the Intro to your Mini __Research Write__ up): **
 * 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.) **
 * Essentiality of this protein: ** Essential in producing, acquiring, and mobilizing inorganic phosphates in the cell signaling transduction pathway
 * Complex of proteins?: ** complexed with othrovanadate (2D1G)
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): ** No evidence showing specific druggability of AcpA in another organism, but there are papers showing druggability of secreted phosphatases in other organisms

http://www.researchgate.net/profile/Yossef_Av-Gay/publication/232529387_Mycobacterium_tuberculosis-secreted_phosphatases_from_pathogenesis_to_targets_for_TB_drug_development/links/0deec5203d3ebdc8f6000000.pdf

[] A discontinuous colorimetric assay was used. Standard 0.2 ml assay mixture contained 0.2 M sodium acetate, pH 6.0, 2.0 mM //p//-nitrophenylphosphate (//p//NPP), and varying amounts of enzyme. Mixtures incubated at 37oC for 15 minutes, and reaction was stopped by addition of 0.2 ml of 0.5 M glycine, pH 10. [] [] __Glycine __
 * *EC#: **3.1.3.2
 * Link to BRENDA EC# page: **
 * -- ** Show screenshot of BRENDA enzyme mechanism schematic
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **
 * -- link to Sigma (or other company ) page for assay (see Sigma links below) **
 * -- -or link (or citation) to paper that contains assay information **
 * -- links to assay reagents (substrates) pages. **
 * --- List cost and quantity of substrate reagents, supplier, and catalog # **

Several different sizes available, see site above for complete list.

 Supplier: //ReagentPlus //®

 10 mg = $17.80

 100 g = $23.50

 1 kg = $64.50

-- PDB # or closest PDB entry if using homology model: 2D1G
 * Structure (PDB or Homology model) **

AgNO3, Arsenate, Cu2+, FeSO4, Hg2+, Molybdate, Orthovanadate, Tartrate, Tungstate, Vanadate, Zn2+ Expressed in //E. coli// BL21 (DE3) SP-Sepharose cation exchange resin (25 ml)
 * Current Inhibitors: **
 * Expression Information (has it been expressed in bacterial cells): **
 * Purification Method : **
 * Image of protein (PyMol with features delineated and shown separately): **

MKLNKITLGILSLSIATTTFATDVNNSKPNDYGTLVKIEQKLFNNANTLKTTTPIKHVVIIFQENNSFDRYFGMYPN AKNPEGEPKFVAKENTPNVNGLTKQLLENNPNTKNPYRLDRNFQPCSQNHEYHQEISSFNGGLMNKFVEHG GHDNDTYKQNCDGQVMGYYDGNTVTALWNYAQNFALNDNTFGTTFGPSTPGALNLVAGANGPAMSPSGNL ENIENNYIIDDPNPYYDDCSYGTSKSGDTNTAVAKITDGYNIGHYLTQKGITWGWFQGGFKPTSYSGKTAICDA MSTNKFGVKSRDYIPHHEPFNYWKETSNPHHLAPSDDKYIGSNDQANHQYDISEFWKALDQNNMPAVSYLKA PGYQDGHGGYSNPLDEQEWLVNTINRIQQSKDWDSTAIIIIYDDSDGDYDHVYSPKSQFSDIKGRQGYGPRLP MLVISPYAKANYVDHSLLNQASVLKFIEYNWGIGSVSKYSNDKYSNNILNMFDFNKEQKTLKLILDPKTGLVMD KLN
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): ( ** NOT INCLUDING ADDED X)
 * *length of your protein in Amino Acids: 514 **

57.78 kDa
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: **

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 91930 Abs 0.1% (=1 g/l) 1.591, assuming all pairs of Cys residues form cystines
 * Molar Extinction coefficient of your protein at 280 nm wavelength: **

Ext. coefficient 91680 Abs 0.1% (=1 g/l) 1.587, assuming all Cys residues are reduced

This present two possible trans-membrane regions and one definite region. Therefore there is 1-3 trans-membrane regions.
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html).

ATGGACGTGAATAATTCTAAGCCGAACGATTACGGTACCCTGGTTAAAATCGAACAGAAACTGTTTAACAA CGCGAATACGCTCAAAACGACCACCCCGATTAAACACGTAGTCATTATCTTCCAGGAGAATAACTCTTTTG ATCGTTACTTCGGTATGTACCCGAATGCGAAGAATCCGGAAGGTGAACCAAAGTTTGTTGCGAAAGAAAA CACCCCAAATGTTAACGGCCTCACCAAACAGCTGCTCGAAAATAACCCGAACACCAAAAATCCGTACCGCC TGGATCGCAACTTCCAGCCGTGCTCTCAGAACCACGAATACCACCAGGAAATCTCTTCTTTCAATGGTGGC CTGATGAATAAGTTCGTCGAACATGGTGGTCACGACAATGACACCTACAAACAGAATTGCGATGGTCAGG TGATGGGCTACTATGATGGCAATACTGTTACCGCGCTGTGGAATTACGCGCAGAACTTCGCGCTGAATGA CAACACCTTTGGTACTACCTTCGGTCCGTCTACCCCGGGTGCCCTGAACCTGGTTGCGGGTGCAAACGGT CCGGCGATGTCTCCGTCTGGCAACCTCGAGAACATCGAAAACAACTACATCATTGACGACCCTAACCCGTA TTATGACGACTGCTCTTACGGCACCTCTAAATCTGGCGACACCAACACTGCGGTTGCTAAGATCACGGATG GTTACAACATCGGTCACTACCTCACCCAAAAAGGTATCACTTGGGGTTGGTTTCAAGGCGGCTTCAAACCG ACCTCTTACTCTGGTAAGACGGCGATTTGTGACGCCATGTCTACCAACAAGTTCGGTGTTAAATCTCGTGA CTATATCCCGCACCACGAGCCTTTCAACTATTGGAAGGAGACCTCCAATCCACACCACCTCGCGCCGAGC GACGACAAATACATTGGTTCTAATGATCAAGCGAATCACCAATACGACATCTCTGAATTCTGGAAGGCGCT CGACCAGAACAACATGCCTGCCGTTTCTTACCTGAAAGCTCCGGGTTACCAGGATGGCCACGGTGGCTAC TCCAACCCACTGGACGAACAGGAATGGCTCGTTAATACCATCAACCGTATCCAGCAGTCTAAAGACTGGG ACTCTACCGCTATTATCATCATTTACGATGACAGCGACGGTGATTACGATCACGTTTACTCTCCAAAGTCTC AATTCTCTGACATCAAGGGCCGTCAAGGTTATGGTCCGCGTCTGCCGATGCTGGTTATCAGCCCGTATGCA AAAGCTAACTACGTTGACCACTCTCTGCTGAATCAGGCGTCTGTCCTGAAATTCATCGAATACAACTGGGG TATCGGTTCCGTATCTAAATACTCTAACGACAAGTATTCTAACAATATCCTGAATATGTTTGACTTCAACAAA GAGCAAAAAACCCTGAAACTGATCCTGGACCCGAAAACTGGTCTCGTTATGGATAAACTGAACTAA
 * *CDS Gene Sequence (paste as text only): **
 * *GC% Content for gene: **47.77%

TACTTCCAATCCATGGACGTGAATAATTCTAAGCCGAACGATTACGGTACCCTGGTTAAAATCGAACAGAAACT GTTTAACAACGCGAATACGCTCAAAACGACCACCCCGATTAAACACGTAGTCATTATCTTCCAGGAGAATAACT CTTTTGATCGTTACTTCGGTATGTACCCGAATGCGAAGAATCCGGAAGGTGAACCAAAGTTTGTTGCGAAAGA AAACACCCCAAATGTTAACGGCCTCACCAAACAGCTGCTCGAAAATAACCCGAACACCAAAAATCCGTACCGC CTGGATCGCAACTTCCAGCCGTGCTCTCAGAACCACGAATACCACCAGGAAATCTCTTCTTTCAATGGTGGCCT GATGAATAAGTTCGTCGAACATGGTGGTCACGACAATGACACCTACAAACAGAATTGCGATGGTCAGGTGAT GGGCTACTATGATGGCAATACTGTTACCGCGCTGTGGAATTACGCGCAGAACTTCGCGCTGAATGACAACAC CTTTGGTACTACCTTCGGTCCGTCTACCCCGGGTGCCCTGAACCTGGTTGCGGGTGCAAACGGTCCGGCGAT GTCTCCGTCTGGCAACCTCGAGAACATCGAAAACAACTACATCATTGACGACCCTAACCCGTATTATGACGACT GCTCTTACGGCACCTCTAAATCTGGCGACACCAACACTGCGGTTGCTAAGATCACGGATGGTTACAACATCGG TCACTACCTCACCCAAAAAGGTATCACTTGGGGTTGGTTTCAAGGCGGCTTCAAACCGACCTCTTACTCTGGTA AGACGGCGATTTGTGACGCCATGTCTACCAACAAGTTCGGTGTTAAATCTCGTGACTATATCCCGCACCACGA GCCTTTCAACTATTGGAAGGAGACCTCCAATCCACACCACCTCGCGCCGAGCGACGACAAATACATTGGTTCT AATGATCAAGCGAATCACCAATACGACATCTCTGAATTCTGGAAGGCGCTCGACCAGAACAACATGCCTGCCG TTTCTTACCTGAAAGCTCCGGGTTACCAGGATGGCCACGGTGGCTACTCCAACCCACTGGACGAACAGGAAT GGCTCGTTAATACCATCAACCGTATCCAGCAGTCTAAAGACTGGGACTCTACCGCTATTATCATCATTTACGAT GACAGCGACGGTGATTACGATCACGTTTACTCTCCAAAGTCTCAATTCTCTGACATCAAGGGCCGTCAAGGTT ATGGTCCGCGTCTGCCGATGCTGGTTATCAGCCCGTATGCAAAAGCTAACTACGTTGACCACTCTCTGCTGAA TCAGGCGTCTGTCCTGAAATTCATCGAATACAACTGGGGTATCGGTTCCGTATCTAAATACTCTAACGACAAGT ATTCTAACAATATCCTGAATATGTTTGACTTCAACAAAGAGCAAAAAACCCTGAAACTGATCCTGGACCCGAAA ACTGGTCTCGTTATGGATAAACTGAACTAACAGTAAAGGTGGATA
 * * **** CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **


 * *GC% Content for gene (codon optimized): **47.65%


 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): ** 38 total

code format="a-c-x-Ha a-c-Ma-td-Ib"  1 ATGGACGTGAATAATTCTAAGCCGAACGATTACGGTACCCTGG  43 2 AGCGTATTCGCGTTGTTAAACAGTTTCTGTTCGATTTTAACCAGGGTACCGTAATCGTTC 60 3 GTTTAACAACGCGAATACGCTCAAAACGACCACCCCGATTAAACACGTAGTCATTATCTT 60 4 ACCGAAGTAACGATCAAAAGAGTTATTCTCCTGGAAGATAATGACTACGTGTTTAATCGG 60 5 ACTCTTTTGATCGTTACTTCGGTATGTACCCGAATGCGAAGAATCCGGAAGGTGAACCAA 60 6 AGGCCGTTAACATTTGGGGTGTTTTCTTTCGCAACAAACTTTGGTTCACCTTCCGGATTC 60 7 CCCCAAATGTTAACGGCCTCACCAAACAGCTGCTCGAAAATAACCCGAACACCAAAAATC 60 8 AGAGCACGGCTGGAAGTTGCGATCCAGGCGGTACGGATTTTTGGTGTTCGGGTTATTTTC 60 9 ACTTCCAGCCGTGCTCTCAGAACCACGAATACCACCAGGAAATCTCTTCTTTCAATGGTG 60 10 CGTGACCACCATGTTCGACGAACTTATTCATCAGGCCACCATTGAAAGAAGAGATTTCCT 60 11 GTCGAACATGGTGGTCACGACAATGACACCTACAAACAGAATTGCGATGGTCAGGTGATG 60 12 TAATTCCACAGCGCGGTAACAGTATTGCCATCATAGTAGCCCATCACCTGACCATCGCAA 60 13 GTTACCGCGCTGTGGAATTACGCGCAGAACTTCGCGCTGAATGACAACACCTTTGGTACT 60 14 ACCAGGTTCAGGGCACCCGGGGTAGACGGACCGAAGGTAGTACCAAAGGTGTTGTCATTC 60 15 GGTGCCCTGAACCTGGTTGCGGGTGCAAACGGTCCGGCGATGTCTCCGTCTGGCAACCTC 60 16 CGGGTTAGGGTCGTCAATGATGTAGTTGTTTTCGATGTTCTCGAGGTTGCCAGACGGAGA 60 17 CATTGACGACCCTAACCCGTATTATGACGACTGCTCTTACGGCACCTCTAAATCTGGCGA 60 18 TGTTGTAACCATCCGTGATCTTAGCAACCGCAGTGTTGGTGTCGCCAGATTTAGAGGTGC 60 19 AGATCACGGATGGTTACAACATCGGTCACTACCTCACCCAAAAAGGTATCACTTGGGGTT 60 20 ACCAGAGTAAGAGGTCGGTTTGAAGCCGCCTTGAAACCAACCCCAAGTGATACCTTTTTG 60 21 AAACCGACCTCTTACTCTGGTAAGACGGCGATTTGTGACGCCATGTCTACCAACAAGTTC 60 22 AGGCTCGTGGTGCGGGATATAGTCACGAGATTTAACACCGAACTTGTTGGTAGACATGGC 60 23 CCGCACCACGAGCCTTTCAACTATTGGAAGGAGACCTCCAATCCACACCACCTCGCGCCG 60 24 ATTGGTGATTCGCTTGATCATTAGAACCAATGTATTTGTCGTCGCTCGGCGCGAGGTGGT 60 25 TAATGATCAAGCGAATCACCAATACGACATCTCTGAATTCTGGAAGGCGCTCGACCAGAA 60 26 CTGGTAACCCGGAGCTTTCAGGTAAGAAACGGCAGGCATGTTGTTCTGGTCGAGCGCCTT 60 27 GAAAGCTCCGGGTTACCAGGATGGCCACGGTGGCTACTCCAACCCACTGGACGAACAGGA 60 28 TCTTTAGACTGCTGGATACGGTTGATGGTATTAACGAGCCATTCCTGTTCGTCCAGTGGG 60 29 CCGTATCCAGCAGTCTAAAGACTGGGACTCTACCGCTATTATCATCATTTACGATGACAG 60 30 CTTTGGAGAGTAAACGTGATCGTAATCACCGTCGCTGTCATCGTAAATGATGATAATAGC 60 31 CGATCACGTTTACTCTCCAAAGTCTCAATTCTCTGACATCAAGGGCCGTCAAGGTTATGG 60 32 GCTTTTGCATACGGGCTGATAACCAGCATCGGCAGACGCGGACCATAACCTTGACGGCCC 60 33 TCAGCCCGTATGCAAAAGCTAACTACGTTGACCACTCTCTGCTGAATCAGGCGTCTGTCC 60 34 AGATACGGAACCGATACCCCAGTTGTATTCGATGAATTTCAGGACAGACGCCTGATTCAG 60 35 GGGGTATCGGTTCCGTATCTAAATACTCTAACGACAAGTATTCTAACAATATCCTGAATA 60 36 CAGGGTTTTTTGCTCTTTGTTGAAGTCAAACATATTCAGGATATTGTTAGAATACTTGTC 60 37 CAACAAAGAGCAAAAAACCCTGAAACTGATCCTGGACCCGAAAACTGGTCTCGTTATGGA 60 38 TTAGTTCAGTTTATCCATAACGAGACCAGTTTTCG 35 code **DNA Works Output:** [|File Link]

__Forward Primer:__ 5’ TAC TTC CAA TCC ATGGAC GTG AAT AAT TCT 3’ 30 bp GC Content 36.7%
 * Primer design results for 'tail' primers (this is just 2 sequences): **

__Reverse Primer__: 5’ TAT CCA CCT TTA CTG TTA GTT CAG TTT ATC CAT 3’ GC Content 33.3%