TargetSp15+-+Acidocalcisomal+exopolyphosphatase+(Leishmania+major+).....................Researcher+-+Edward+S.


 * *Target (protein/gene name): **** acidocalcisomal exopolyphosphatase **
 * *NCBI Gene # or RefSeq#: **** LMJF_01_0310 **


 * *Protein ID (NP or XP #) or Wolbachia#: **** 29931 **


 * *Organism (including strain): **** Leishmania major strain Friedlin **


 * Etiologic Risk Group (see link below): **** N/A **


 * /**Disease Information** (sort of like the Intro to your Mini Research Write up):
 * Sometimes mistaken with leprosy, the affliction known as leishmaniosis, causes skin ulcers to erupt and may also lead to an enlarged liver and spleen – eventually leading to anemia. The disease is spread by the bite of sandflies, but the real culprit is the parasite protozoon belonging to the genus //Leishmania//; there are many species of this genus that cause the disease. Currently, about 12 million people are affected, with 20 to 50 thousand deaths per year. **


 * Different species need to be treated differently. For examples, //Leishmania major//, is resistant to the oral medication Miltefosine; however, Pentamidine is effective. **


 * Link to TDR Targets page (if present): ** http://www.tdrtargets.org/targets/view?gene_id=29931


 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) **

**Essentiality of this protein: http://www.ncbi.nlm.nih.gov/gene/12983123 **

Is it a monomer or multimer as biological unit**? (make prediction at** http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): multimer


 * Complex of proteins?: **** Monomer (chains a and b) **


 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **** N/A **

***EC#: 3.6.1.11 **

**Link to BRENDA EC# page:**** http://www.brenda-enzymes.org/enzyme.php?ecno=3.6.1.11 **

**--** 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) **** http://www.sigmaaldrich.com/catalog/papers/12393865 **


 * --- List cost and quantity of substrate reagents, supplier, and catalog # **

Fetal bovine serum: Cost must be inquired, Sigma, F2442 Dulbecco’s PBS: $15.40 for 100ML, Sigma, D1408 Protease inhibitor mixture: $64.10 for 1ML, Sigma, P8849 Protease inhibitor mixture: 71.80 for 1ML, Sigma, P8340 Ampicillin: $63.80 for 5G, Sigma, A9393 Kanamycin: $56.30 for 20ML, Sigma, K0254 Paraformaldehyde: $34.80 for 500G, Sigma, P6148 Glutaraldehyde: $116.50 for 10X1ML, Sigma, G5882 Bovine serum albumin: $276.00 for 50ML, Sigma, A7979 ATP: $293.50 for 1VL, Sigma, FLAAM PPi: $56.40 for 1VL, Sigma, P7275 phosphate glass: $121.50 for 500MG, Aldrich, S4379 proteinase K: $39.50 for 5MG, Aldrich, P2308 lysozyme: $49.50 for 1G, Sigma, L6876 horseradish peroxidase-conjugated anti-rabbit: $403.50 for 200UL, Sigma, H0912 fluorescein-conjugated goat anti-rabbit: $125.00 for 1ML, Sigma, F6005 rhodamine-conjugated goat anti-mouse IgGs: $178.50 for 1ML, Sigma, A0168

**Structure (PDB or Homology model)**

-- PDB # or closest PDB entry if using homology model: 4ITY

-- For Homology Model option:

Show pairwise alignment of your BLASTP search in NCBI against the PDB

Query Coverage: 99%

Max % Identities: 93%

Max Score: 1700

Total Score: 1700

**Current Inhibitors: None **

**Expression Information (has it been expressed in bacterial cells): Expressed in //Salmonella enterica, E. coli.// **

**Purification Method** **:** From: http://www.sciencedirect.com/science/article/pii/S0014579398005912 The vacuolar fraction free of other cell organelles [|[8]] was frozen at −20°C in 10 mM Tris-HCl, pH 7.2, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride and after thawing treated with ultrasound (MSE, USA) for 10 s. Vacuolar membranes were sedimented at 15 000×//g// for 90 min. The supernatant was applied to a Q-Sepharose column (1.6×5 cm) equilibrated with 25 mM Tris-acetate, pH 7.2, containing 0.1% Triton X-100 as enzyme stabilizer. After washing with the same buffer, extraneous proteins were eluted with the same buffer, containing 0.3 M KCl. PolyPase was eluted with 0.7 M KCl in the same buffer. Removal of KCl and substitution of the buffer with 25 mM Tris-acetate, pH 6.0, was performed in an Amicon system (PM-10 membrane). Thereafter, the preparation was applied to an S-Sepharose column (1.6×5 cm) equilibrated with 25 mM Tris-acetate, pH 6.0, containing 0.1% Triton X-100. Extraneous proteins were eluted with the same buffer, containing 0.2 M KCl. PolyPase was eluted with increasing concentrations of KCl (0.2–1.2 M) in the same buffer. The enzyme preparation was stored at −20°C.

***Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**
 * Image of protein (PyMol with features delineated and shown separately): **

code 1__0__        2__0__         3__0__         4__0__         5__0__         6__0__

MSGVINDFLR RCLKKVAGKV QPLTVVQGNE GGDMDSIVGC IYLAMLFDKQ PKFGFENPVP

7__0__        8__0__         9__0__        10__0__        11__0__        12__0__

ALNFPQEDFG LRNDVTNLFK ELGIDASLLM SVQRGQIAHN LVDIAALNAS VVLYDHNKLR

13__0__       14__0__        15__0__        16__0__        17__0__        18__0__

ENQSDLASRV VGVVDHHFDE QQYLKTASKL RVLRTVGSAC TLVTELYREC GEDVVCPTLL

19__0__       20__0__        21__0__        22__0__        23__0__        24__0__

TAPIVLDTVN FEPAQKKVTP EDIAAYEWLR AKEVADSADA AALFEKLSKW KDDVLALSVP

25__0__       26__0__        27__0__        28__0__        29__0__        30__0__

QILRRDYKQF SFKARTQKGV MSAGTSSVPC ACKQLEAHFS VDLIVAEAAK YVEQHQLDVL

31__0__       32__0__        33__0__        34__0__        35__0__        36__0__

IVAFAGKVGG KHTREVAFCA KPDVISFFAP FVAEAPDGVS FTVITKCQTV DGSYEYASYS

37__0__       38__0__

LSDPSISRKK LVPALSEFLA EGTRSLCE code


 * *length of your protein in Amino Acids: **** 388 Amino Acids **

** Molecular Weight ** ** of your protein in kiloDaltons using the ** [|**Expasy ProtParam**] ** website: ** 42.5948 kilo Daltons

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 26525 Abs 0.1% (=1 g/l) 0.623, assuming all pairs of Cys residues form cystines Ext. coefficient 25900 Abs 0.1% (=1 g/l) 0.608, assuming all Cys residues are reduced
 * Molar ** ** Extinction coefficient ** ** of your protein at 280 nm wavelength: **


 * TMpred graph Image ** (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

ATGTCTGGCGTTATCAACGACTTCTTGCGCCGCTGCCTCAAGAAGGTTGCCGGCAAAGTGCAGCCACTGA CCGTGGTGCAGGGAAACGAGGGAGGTGACATGGACAGCATTGTCGGCTGCATTTACCTCGCCATGCTTTT TGATAAGCAGCCCAAATTCGGTTTTGAGAACCCTGTGCCGGCGCTGAACTTTCCACAAGAAGATTTCGGC CTGCGCAACGATGTGACGAATCTCTTCAAGGAGCTCGGGATCGATGCATCGCTCTTGATGTCGGTACAGA GGGGACAGATCGCGCATAACCTTGTCGATATTGCTGCTCTCAACGCTTCGGTTGTTCTTTATGATCACAA TAAGCTTAGGGAGAACCAAAGCGACCTCGCCTCTAGAGTTGTCGGAGTAGTGGACCATCACTTTGACGAG CAGCAGTACCTCAAAACCGCATCCAAGCTAAGAGTTCTGCGAACTGTCGGATCCGCGTGCACACTCGTTA CGGAGCTCTACCGCGAATGTGGCGAGGATGTTGTGTGTCCCACGCTGCTAACGGCTCCGATTGTTCTGGA TACAGTGAACTTTGAGCCGGCACAAAAGAAGGTGACACCTGAGGATATCGCTGCATACGAGTGGTTGCGT GCAAAGGAGGTCGCTGACAGTGCTGACGCCGCGGCTCTCTTTGAAAAGCTGTCGAAGTGGAAGGATGACG TGCTGGCACTCAGCGTTCCACAAATCTTGAGGCGAGATTACAAACAGTTCAGCTTCAAAGCCAGAACGCA GAAGGGCGTTATGTCCGCGGGCACCAGTAGTGTGCCGTGTGCGTGCAAGCAGCTAGAAGCTCACTTTTCT GTGGATTTAATTGTGGCGGAGGCTGCCAAGTACGTGGAGCAGCATCAGTTGGATGTGCTCATCGTTGCAT TTGCCGGGAAAGTCGGCGGCAAGCACACTCGCGAGGTCGCCTTCTGTGCAAAGCCTGACGTGATCTCCTT TTTTGCTCCTTTTGTAGCGGAGGCCCCGGATGGCGTGTCATTTACCGTGATCACAAAGTGTCAGACGGTG GACGGATCATACGAGTACGCCTCGTACTCCCTCAGCGACCCTTCTATTTCTCGCAAGAAGCTGGTTCCAG CTCTGTCCGAATTCCTTGCCGAGGGGACACGGAGTCTTTGTGAGTAA
 * *CDS ** ** Gene Sequence ** ** (paste as text only): **


 * *GC% Content for gene: N/A **


 * *CDS Gene Sequence (codon optimized) - copy from ** ** output ** ** of ** ** Primer Design ** ** Protocol (paste as text only): N/A **


 * *GC% Content for gene (codon optimized): N/A **