Target+-+Putative+acid+phosphatase+(Helicobacter+pylori)

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and plays important roles in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori has been found in the stomachs of humans in all parts of the world.70 to 90% of the population carries H. pylori in developing countries, while in developed countries, the percentage of infection is much lower. Interestingly, H. pylori's perfect habitat in human is in the stomach, where strong stomach acid is. Transmission of H. pylori can occur by iatrogenic, meaning it is trasmitted during medical treatment, fecal-oral, and oral-oral routes. H. pylori is able to colonize and persist in a unique biological niche within the gastric lumen. A variety of tests to diagnose H. pylori infection are now available. Histological examination of gastric tissue, culture, rapid urease testing, DNA probes, and PCR analysis, when used to test gastric tissue, all require endoscopy. In contrast, breath tests, serology, gastric juice PCR, and urinary excretion of ammonia are noninvasive tests that do not require endoscopy.
 * Target (protein/gene name):** putative acid phosphatase
 * NCBI Gene # or RefSeq#:** 12364462
 * Protein ID (NP or XP #) or Wolbachia#:** YP_005784253.1
 * Organism (including strain):** //Helicobacter pylori 2017//
 * Etiologic Risk Group (see link below):** Risk group I
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**

Reference: Dunn, B. E.; Cohen, H.; Blaser, M. J., Helicobacter pylori. // Clin Microbiol Rev //** 1997, ** // 10 // (4), 720-41.


 * Essentiality of this protein:**
 * Complex of proteins?:**
 * Druggable Target:**

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 * *EC#: **EC 3.1.3.2
 * Link to BRENDA EC# page:**
 * --** Show screenshot of BRENDA enzyme mechanism schematic

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 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**
 * -- 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**
 * A. 90 mM Citrate Buffer Solution, pH 4.8 at 37°C
 * (Use Citrate Buffer Solution, Sigma Stock No. 104-4.)
 * $47.00
 * B. 40 mM Tartrate Buffer, with 90 mM Citrate, pH 4.8 at 37°C.
 * (Use Tartrate Acid Buffer Solution, Sigma Stock No. 104-12.)
 * $37.30
 * C. 11.3 mM p-Nitrophenyl Phosphate (PNPP)
 * (Prepare 5 ml in deionized water using p-Nitrophenyl Phosphate Sigma 104 Phosphatase Substrate, Sigma Stock No. 104-0.)
 * $79.90
 * D. 100 mM Sodium Hydroxide Solution (NaOH)
 * (Prepare 50 ml in deionized water using Sodium Hydroxide, Anhydrous Sigma Prod.No. S-5881.)
 * $27.30
 * E. Prostatic Acid Phosphatase Enzyme Solution
 * (Immediately before use, prepare a solution containing 0.015 - 0.02 unit/ml of Acid Phosphatase in cold deionized water.)

-- PDB # or closest PDB entry if using homology model: **2I33** (homologous model) -- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB
 * Structure Available (PDB or Homology model)**

Query Coverage: Max % Identities: **40%** % Positives: **58%** Chain used for homology: **A**

- orthovanadate - N/A All purification steps were carried out at 4 degress Celcius. Buffers: A, 100 mM HEPES-Tris (pH 8.0) supplemented with 100mM KCl, 10% glycerol, 1% n-octyl β-D-glucopyranoside (OGP), 1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM MgCl2, and 0.5 mM phenylmethanesulfonyl fluoride (PMSF); B, 20 mM HEPES-Tris (pH 8.0) containing 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, and 0.1% OGP. H. pylori membrane fractions containing both inner and outer membranes were dissolved in buffer A. After incubating for 2 h with gentle stirring, the insoluble factions were removed by centrifugation (10,000 × g, 10min). The supernatant was then collected as the solubilized membrane fractions. The OGP extract was <span style="font-family: Arial,Helvetica,sans-serif;">concentrated and then chromatographed on a DEAE-Sepharose CLB <span style="font-family: Arial,Helvetica,sans-serif;">column that was previously equilibrated with buffer B. After <span style="font-family: Arial,Helvetica,sans-serif;">washing thoroughly with buffer B, the bound proteins were eluted <span style="font-family: Arial,Helvetica,sans-serif;">with a linear KCl gradient (0-1M) and fractionated. Fractions <span style="font-family: Arial,Helvetica,sans-serif;">containing ATPase activity that was increased in the presence 100mM <span style="font-family: Arial,Helvetica,sans-serif;">NH4Cl were pooled and dialyzed against 1M ammonium sulfate <span style="font-family: Arial,Helvetica,sans-serif;">with buffer B. The resulting sample was loaded onto a Phenyl- <span style="font-family: Arial,Helvetica,sans-serif;">Sepharose column that was pre-equilibrated with buffer B containing <span style="font-family: Arial,Helvetica,sans-serif;">1M ammonium sulfate and thoroughly washed with the same <span style="font-family: Arial,Helvetica,sans-serif;">buffer. The bound proteins were eluted using an ammonium-sulfated <span style="font-family: Arial,Helvetica,sans-serif;">gradient (a linear 1M to 0M) and fractionated. The active fractions <span style="font-family: Arial,Helvetica,sans-serif;">were concentrated using a Centricon-YM 10 (Amicon).
 * Current Inhibitors:**
 * Expression Information (has it been expressed in bacterial cells):**
 * Purification Method:**

<span style="font-family: Arial,Helvetica,sans-serif;">Reference: Ki, M. R.; Yun, S. K.; Choi, K. M.; Hwang, S. Y., Identification and characterization of the acid phosphatase HppA in Helicobacter pylori. // J Microbiol Biotechnol //** 2011, ** // 21 // (5), 483-93.

code gi|385224327|ref|YP_005784253.1| putative acid phosphatase [Helicobacter pylori 2017] <span class="ff_line">MVKKTLASVLLGLSLMSVLDAKECVSPITRSVKYHQQSAEIRALQLQSYKMAKMALDNNLKLVKDKKPAV ILDLDETVLNTFDYAGYLVKNCIKYTPETWDKFEKEGSLTLIPGALDFLEYANSKGVKIFYISNRTQKNK AFTLKTLKSFKLPQVSEESVLLKEKGKPKAVRRELVAKDYAIVLQVGDTLHDFDAIFAKDAKNSQEQQAK VLQNAQKFGTEWIILPNSLYGTWEDEPIKAWQNKK code
 * Image of protein (PyMol with features delineated and shown separately):**
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**

code gi|385223048:c1263962-1263225 Helicobacter pylori 2017 chromosome, complete genome <span class="ff_line">ATGGTAAAAAAGACCCTTGCATCGGTTTTATTAGGATTGAGTTTGATGAGTGTGTTAGACGCTAAAGAGT GCGTTTCGCCGATAACAAGAAGCGTTAAGTATCATCAGCAAAGCGCTGAGATCAGAGCCTTGCAATTGCA AAGTTACAAAATGGCAAAAATGGCGCTAGACAATAATCTCAAGCTCGTTAAAGACAAAAAGCCAGCTGTC ATCTTGGATTTAGATGAAACCGTTTTGAACACTTTTGATTATGCGGGCTATTTGGTCAAAAATTGCATTA AATACACCCCAGAAACTTGGGATAAATTTGAAAAAGAAGGCTCTCTTACGCTCATTCCTGGAGCGCTAGA CTTTTTAGAATACGCTAATTCTAAGGGCGTTAAGATTTTTTACATTTCTAACCGCACCCAAAAAAATAAG GCATTCACTTTAAAAACGCTCAAAAGTTTCAAACTCCCCCAAGTGAGTGAAGAATCCGTTTTATTAAAAG AAAAAGGTAAGCCTAAAGCCGTTAGGCGGGAATTAGTCGCTAAGGATTATGCGATTGTTTTACAAGTGGG CGACACTTTGCATGATTTTGACGCTATTTTTGCTAAAGACGCTAAAAACAGCCAAGAACAACAAGCCAAA GTCTTGCAAAACGCTCAAAAATTCGGCACAGAATGGATCATTTTACCCAACTCTCTTTATGGCACATGGG AAGATGAGCCTATAAAAGCATGGCAAAATAAAAAATAA code
 * length of your protein in Amino Acids:** 245
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website:** 27811.3 g/mol
 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 35535 M^-1 cm^-1
 * 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):**
 * GC% Content for gene:** 50%
 * 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) -- 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 pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):**
 * ( link to DNA Works output text file - **that should be saved in your Google Docs folder after you did the primer design protocol)


 * Primer design results for 'tail' primers (this is just 2 sequences):**