Target-protein+tyrosine+phosphatase+(Entamoeba+hystolytica)


 * *Target (protein/gene name): **Protein Tyrosine Phosphatase
 * *NCBI Gene # or RefSeq#: ** had a hard time finding when searching backwards from protein ID
 * *Protein ID (NP or XP #) or Wolbachia#: ** XM_001914124.1

Entamoeba histolytica is an anaerobic parasitic protozoan that causes amoebic colitis and amoebiasis, the second leading cause of death from a parasitic disease in the world. The protozoan is transmitted in fecal matter through direct contact or food/water contamination. When the organism reaches the intestines, it secretes the phosphatase, which then proceeds to catalyze the p-nitrophenyl phosphate (pNPP), leading to hydrolysis at acidic pH levels. This kills host cells on contact and leads to tissue death. It is also possible that the organism can travel to the liver and cause abscesses in the organ. Symptoms include diarrhea or dysentery, fatigue, weight loss, fever, and abdominal pain. Some antibiotics can be used in the case of a liver abscess but there is no vaccine for amoebiasis in general. Prevention can be taken in the form of food, water, and personal hygiene. The disease occurs mostly in areas with poor sanitation around the world. @https://www.neb.com/tools-and-resources/feature-articles/parasitic-infections-in-humans @http://www.sciencedirect.com/science/article/pii/S002075190800146X @http://iai.asm.org/content/70/4/1816.short @http://www.sciencedirect.com/science/article/pii/S0020751903000298
 * *Organism (including strain): **Entamoeba histolytica HM-1: IMSS
 * Etiologic Risk Group (see link below): **Appendix B-II-C. Risk Group 2 (RG2)-Parasitic Agents
 * *Background/Disease Information (sort of like the Intro to your Mini Research Write up): **


 * Essentiality of this protein: **The protein is thought to be a key factor in causing host cell death.
 * Complex of proteins?: **Just secreted phosphotyrosine phosphatas** e **
 * Druggable Target: **Research is currently validating the essentiality of the protein, and shows the ability to potentially inhibit it.

Figure 1: Mechanism schematic for protein tyrosine phosphatase.
 * *EC#: ** 3.1.3.48
 * Link to BRENDA EC# page: ** []

[] -pNPP assay: $314, Sigma, 1000 well assays/100 tube assays
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **Previous research measured using a spectrophotometer (405 nm). Used pNPP, NaOH, and //o-//phospho//-l-//tyrosine (P-Tyr)
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates): **
 * -- link (or citation) to paper that contains assay information: ** []
 * -- List cost and quantity of substrate reagents and supplier **
 * - **// o- // phospho // -l- // tyrosine (P-Tyr): $76.20, Sigma, 250 mg

**Structure Available (PDB or Homology model) **

*Solved structures available: 3IDO, 3ILY, 3JS5, 3JVI
-- PDB # or closest PDB entry if using homology model: 3JS5 -- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB Figure 2: Side-by-side BLASTP comparison of 3JS5 and human phosphotyrosyl phosphotase.

Query Coverage:76% Max % Identities:36% % Positives: 52% Chain used for homology: Chain A, Crystal Structure of A Human Low Molecular Weight Phosphotyrosyl Phosphatase. Implications for Substrate Specificity.

C. This was then diluted with an equilibration buffer and passed through a purification column (Con A-Sepharose). The acid phosphatase was then eluted with a buffer (0.5 M α- methyl-d-mannoside). Samples that showed enzyme activity were passed through another column (DEAE-Cellulose), washed with an equilibration buffer, and eluted with a sodium chloride gradient. []) Figure 3: Crystal structure of protein tyrosine phosphatase from Entamoeba histolytica with Hepes in the active site.  []
 * Current Inhibitors: ** Ammonium molybdate, sodium tungstate, sodium //o//-vanadate
 * Expression Information (has it been expressed in bacterial cells): **It has been expressed in humans, rats, cattle, and yeast. Cells have been cultured in TYI-S-33.
 * Purification Method: ** E. histolytica was grown in medium, then the excretory phosphatase was separated from the supernatant. The supernatants were concentrated using a pressure ultrafiltration (Amicon PM 30 membrane) and stirred cell apparatus at 4 °
 * Image of protein (PyMol with features delineated and shown separately): **

mahhhhhhmgtleaqtqgpgs mkllfvclgnicrspaaeavmkkviqnhhltekyicdsagtcsyhegqqadsrmrkvgksrgyqvdsisrpvvss dfknfdyifamdndnyyelldrcpeqykqkifkmvdfcttikttevpdpyyggekgfhrvidiledacenliikleegklin Of shortened sequence: code Ext. coefficient   13785 Abs 0.1% (=1 g/l)  0.763, assuming all pairs of Cys residues form cystines
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **
 * *length of your protein in Amino Acids: ** 178......... 157
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: ** 20351.1........... 18067.7
 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 13785

Ext. coefficient   13410 Abs 0.1% (=1 g/l)  0.742, assuming all Cys residues are reduced code

ATGAAACTGCTGTTCGTTTGCCTGGGTAACATCTGCCGTTCTCCGGCTGCGGAAGCGGTT ATGAAAAAAGTTATCCAGAACCACCACCTGACCGAAAAATACATCTGTGACTCTGCGGGT ACCTGCTCTTACCACGAAGGTCAGCAGGCGGACTCTCGTATGCGTAAAGTTGGTAAATCT CGTGGTTACCAGGTTGACTCTATCTCTCGTCCGGTTGTTTCTTCTGACTTCAAGAACTTT GACTACATCTTCGCGATGGACAACGACAACTACTACGAACTCCTGGACCGTTGCCCGGAA CAGTACAAACAGAAAATCTTCAAAATGGTAGACTTCTGCACCACCATCAAAACCACCGAA GTTCCGGACCCGTACTACGGTGGTGAAAAAGGTTTCCACCGTGTTATCGACATCCTGGAG GACGCGTGCGAAAACCTGATCATCAAACTGGAAGAAGGTAAACTGATCAACTAA ***GC% Content for gene:** 47.89% TACTTCCAATCC __ATG ____<span style="font-family: &#39;Courier New&#39;; font-size: 13.3333px;">AAACTGCTGTTCGTT __<span style="font-family: &#39;Courier New&#39;; font-size: 13.3333px;">TGCCTGGGTAACATCTGCCGTTCTCCGGCTGCGGAAGCGGTT <span style="font-family: &#39;Courier New&#39;; font-size: 13.3333px;">ATGAAAAAAGTTATCCAGAACCACCACCTGACCGAAAAATACATCTGTGACTCTGCGGGT <span style="font-family: &#39;Courier New&#39;; font-size: 13.3333px;">ACCTGCTCTTACCACGAAGGTCAGCAGGCGGACTCTCGTATGCGTAAAGTTGGTAAATCT <span style="font-family: &#39;Courier New&#39;; font-size: 13.3333px;">CGTGGTTACCAGGTTGACTCTATCTCTCGTCCGGTTGTTTCTTCTGACTTCAAGAACTTT <span style="font-family: &#39;Courier New&#39;; font-size: 13.3333px;">GACTACATCTTCGCGATGGACAACGACAACTACTACGAACTCCTGGACCGTTGCCCGGAA <span style="font-family: &#39;Courier New&#39;; font-size: 13.3333px;">CAGTACAAACAGAAAATCTTCAAAATGGTAGACTTCTGCACCACCATCAAAACCACCGAA <span style="font-family: &#39;Courier New&#39;; font-size: 13.3333px;">GTTCCGGACCCGTACTACGGTGGTGAAAAAGGTTTCCACCGTGTTATCGACATCCTGGAG GACGCGTGCGAAAACCTGATCATCAAACTGGAA__GAAGGTAAACTGATCAACTAA__CAGTAAAGGTGGATA
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * *CDS Gene Sequence (paste as text only): **
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **
 * NOTE - this seems to have the 'tail' on the beginning and end for cloning. So, start at ATG and end at TAA **
 * *GC% Content for gene (codon optimized): ** 47.5 ** % **

code format="drive-viewer-text-page" The DNA sequence #  1 is: 1 ATGAAACTGCTGTTCGTTTGCCTGGGTAACATCTGCCGTTCTCCGGCTGCGGAAGCGGTT 61 ATGAAAAAAGTTATCCAGAACCACCACCTGACCGAAAAATACATCTGTGACTCTGCGGGT 121 ACCTGCTCTTACCACGAAGGTCAGCAGGCGGACTCTCGTATGCGTAAAGTTGGTAAATCT 181 CGTGGTTACCAGGTTGACTCTATCTCTCGTCCGGTTGTTTCTTCTGACTTCAAGAACTTT 241 GACTACATCTTCGCGATGGACAACGACAACTACTACGAACTCCTGGACCGTTGCCCGGAA 301 CAGTACAAACAGAAAATCTTCAAAATGGTAGACTTCTGCACCACCATCAAAACCACCGAA 361 GTTCCGGACCCGTACTACGGTGGTGAAAAAGGTTTCCACCGTGTTATCGACATCCTGGAG 421 GACGCGTGCGAAAACCTGATCATCAAACTGGAAGAAGGTAAACTGATCAACTAA code
 * CODON OPTIMIZED SEQUENCE from **


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

__<span style="font-family: &#39;Courier New&#39;; font-size: 10pt;">Forward Primer: __ <span style="font-family: &#39;Times New Roman&#39;,serif; font-size: 10pt;"> ’ TACTTCCAATCC ** ATG **__<span style="font-family: &#39;Courier New&#39;; font-size: 10pt;">AAACTGCTGTTCG __<span style="font-family: &#39;Courier New&#39;; font-size: 10pt;"> ’ __28__ bp
 * Primer design results for 'tail' primers (this is just 2 sequences): **

__<span style="font-family: &#39;Courier New&#39;; font-size: 10pt;">Reverse Primer __<span style="font-family: &#39;Courier New&#39;; font-size: 10pt;">: ’ __GGTAAACTGATCAAC** TAA **__ CAGTAAAGGTGGATA ' __<span style="font-family: &#39;Courier New&#39;;">36 __<span style="font-family: &#39;Courier New&#39;;"> bp

**Resources:** See ** ProtocolTargetDiscoveryVDS.docx ** for more Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334 [] [] @http://www.niaid.nih.gov/Pages/default.aspx @http://eupathdb.org/eupathdb/ @https://patricbrc.vbi.vt.edu/portal/portal/patric/Home @http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes @http://tubic.tju.edu.cn/deg/ @http://csgid.org/csgid/cake/pages/community_request_gateway @http://tdrtargets.org/ @http://gsc.jcvi.org/status.shtml @http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm Genome: http://www.ncbi.nlm.nih.gov/nuccore/183235131?report=fasta NCBI GENE Page: @http://www.ncbi.nlm.nih.gov/gene BLAST Page: @http://blast.ncbi.nlm.nih.gov/ Protein Sequence: http://www.ncbi.nlm.nih.gov/protein/472460682?report=fasta NCBI Protein Page: @http://www.ncbi.nlm.nih.gov/protein Protein Expression Website Protein Expression Paper:
 * Scientific Journals: **
 * Databases of genes/organisms: **
 * Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations) **
 * Gene Information: **
 * Protein Information: **
 * Primer Overlap PCR Articles **
 * Is my target good for Virtual Screening programs? **