Target-Protein+Tyrosine+Phosphatase+(Listeria+monocytogenes)

//L. monocytogenes// is a food-borne bacterium that leads to listeriosis; it is the third most common foodborne bacterial pathogen. It can be found in raw vegetables, meat, raw or unpasteurized milk, and dairy products containing such milk. Those at greatest risk include newborn babies (in which listeriosis often results in sepsis and/or meningitis), pregnant mothers, elderly, and those with weakened immune systems, such as HIV patients. Symptoms include nausea, fever, and diarrhea, with a mortality rate of 20-30%. Its fatality rates can surpass those of //Salmonella//. As a Gram-positive facultative anaerobe, //L. monocytogenes// is able to survive in both aerobic and anaerobic environments. It expresses the protein beta hemolysin, and is thus able to destroy the host's red blood cells. //L. monocytogenes// contains D-galactose residues on its surface that interact with D-galactose receptors of the host's intestinal cells, allowing the bacteria to move past the intestinal walls into the host's body. Another protein, internalin, also interacts with intestinal cell proteins to allow entry and invasion. Once it has invaded, the bacterium moves swiftly through the host cell and multiplies, often passing through the blood-brain barrier. Is it a monomer or multimer as biological unit ** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): N/A Figure 2 - Brenda Reaction Mechanism for Tyrosine Phosphatase protein (EC # 3.1.3.48) from http://www.brenda-enzymes.org/structure.php?show=reaction&id=11748&type=I&displayType=marvin
 * Target Team: ** Monica L., Tyler T., James F.
 * *Target (protein/gene name): **Tyrosine Phosphatase
 * *NCBI Gene # or RefSeq#: ** Gene #: 985934
 * *Protein ID (NP or XP #) or Wolbachia#: ** NP_465325.1
 * *Organism (including strain): **//Listeria monocytogenes//
 * Etiologic Risk Group (see link below): **Risk Group 2 (moderate individual risk, low community risk)
 * */ 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.): ** http://www.ncbi.nlm.nih.gov/gene/?term=985934
 * Essentiality of this protein: ** http://iai.asm.org/content/79/6/2489.full
 * Complex of proteins?: ** N/A
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): ** http://www.jbc.org/content/279/23/24226.long
 * *EC#: **3.1.3.48
 * Link to BRENDA EC# page: **http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.48
 * -- ** Show screenshot of BRENDA enzyme mechanism schematic

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/larprtntyrosinephos.pdf
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **Spectrophotometric Stop Rate Determination
 * -- link to Sigma (or other company ) page for assay (see Sigma links below) **
 * -- -or link (or citation) to paper that contains assay information **

__Imidazole:__[] __Phosphatase Substrate:__[] __Sodium Chloride:__[] __DL-Dithiothreitol:__[] __Ethylenediaminetetraacetic acid disodium salt dehydrate:__[] __Bovine Serum Albumin:__[] __Sodium Hydroxide:__[]
 * -- links to assay reagents (substrates) pages. **


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

__**All reagents are from Sigma-Aldrich:**__ __Imidazole:__ I202-1G ($11.50) = 1 gram __Phosphatase Substrate:__ S0942 ($35.50) = 50 Tablets __Sodium Chloride:__ S9625 ($33.90) = 500 grams __DL-Dithiothreitol:__ D0632 ($41.70) = 1 gram __Ethylenediaminetetraacetic acid disodium salt dehydrate:__ ED2SS ($25.70) = 50 grams __Bovine Serum Albumin:__ A8022 ($66.90) = 10 grams __Sodium Hydroxide:__ S5881 ($51.20) = 500 grams -- PDB # or closest PDB entry if using homology model: Not available (will derive homology model later on) -- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB Figure 3 - BLASTP pairwise alignment between protein tyrosine phosphate in // Listeria monocytogenes // and 1YWF
 * Structure (PDB or Homology model) **

Query Coverage: 84% Max % Identities: 26% % Positives: 47% Chain used for homology: A

SSEVATKPDPKLTDVDYTHDSVMKDNGTSTSTQDLTASLAKMDNPETFLINANKSFITDETSIQAYKDFFDILLANQDGSVLWHCTAGKDRAGFGTALVLSALGV DKNTVIDDYMLSNKYRADENKKAIEAVAAKTDNKKVIDGMTAVMEVRESYINAAFDEINAKYGSMDNFLKEKLGLTDAKKEQLKKAYLY Figure 4 - TMpred graph for lmptp
 * Current Inhibitors: ** N/A
 * Expression Information (has it been expressed in bacterial cells): ** http://www.biomedcentral.com/1471-2164/11/457
 * Purification Method : ** Glutathione sepharose affinity chromatography
 * Image of protein (PyMol with features delineated and shown separately): ** N/A
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **MKNWVKVTGAGVLSATLLLGGCGAQSEEKAEANVKTEQTLKPGSQIKLEGAVNVRDLGGYKTTDGLTIKPHKLIRSAELANLSDSDKKKLVNTYDLSHIVDFRT
 * *length of your protein in Amino Acids: ** 298
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: ** 32585.8
 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 26025
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

ATGAAAAATTGGGTAAAAGTAACAGGAGCAGGGGTACTAAGCGCGACTTTACTATTAGGTGGATGCGGGG CGCAGTCAGAAGAAAAAGCAGAAGCAAATGTTAAAACCGAGCAAACACTCAAACCAGGAAGCCAAATTAA ATTAGAAGGCGCTGTAAATGTCCGGGACTTAGGCGGATACAAAACAACGGATGGACTAACCATTAAGCCA CATAAACTCATTAGAAGTGCCGAACTCGCTAACTTAAGTGATTCGGATAAAAAGAAACTCGTAAATACAT ATGATTTGTCTCATATAGTTGATTTCCGAACAAGTTCAGAAGTCGCAACGAAACCAGATCCGAAACTCAC AGATGTAGACTATACGCACGATTCTGTGATGAAAGATAATGGAACATCTACAAGCACACAAGATTTAACT GCTAGCCTAGCCAAAATGGATAATCCAGAGACATTTCTCATTAATGCTAATAAGAGCTTTATTACAGATG AAACTTCGATACAAGCCTATAAAGATTTTTTTGATATACTACTAGCAAACCAAGATGGTTCTGTTCTTTG GCACTGTACAGCTGGAAAAGACCGAGCTGGATTTGGAACCGCCCTCGTTCTTTCAGCGTTAGGTGTGGAT AAAAACACGGTCATTGACGATTATATGCTGTCCAATAAATATCGTGCTGACGAAAATAAAAAAGCAATTG AAGCCGTTGCAGCAAAAACAGATAATAAAAAAGTGATTGATGGAATGACAGCCGTAATGGAAGTTCGTGA ATCTTATATCAATGCAGCCTTCGATGAAATTAATGCAAAATATGGTTCGATGGACAACTTTTTAAAAGAA AAACTCGGACTAACCGATGCTAAAAAAGAACAACTAAAAAAAGCATATCTTTATTAA
 * *CDS Gen ****  e Sequence (paste as text only): **


 * *GC% Content for gene: ** 37.9%

1 ATGAAAAACTGGGTTAAGGTTACCGGTGCGGGTGTTCTCTCTGCGACCCTGCTCCTGGGC 61 GGCTGCGGTGCGCAGTCTGAAGAGAAGGCTGAAGCGAACGTCAAAACCGAACAGACCCTG 121 AAACCGGGCTCTCAGATCAAACTGGAAGGTGCGGTAAACGTTCGTGATCTGGGTGGCTAC 181 AAGACCACCGACGGCCTGACCATCAAGCCGCACAAACTGATCCGTTCTGCGGAACTCGCG 241 AATCTGAGCGACTCTGACAAGAAGAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 301 GACTTCCGTACCTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTAGAC 361 TATACCCACGATAGCGTTATGAAGGACAACGGTACCAGCACGTCTACCCAGGATCTGACC 421 GCCTCTCTGGCTAAAATGGATAACCCGGAAACCTTCCTGATCAACGCAAACAAGTCTTTC 481 ATTACTGACGAAACGTCTATCCAGGCGTACAAGGACTTCTTCGACATCCTGCTGGCGAAC 541 CAGGACGGTTCTGTTCTGTGGCACTGCACGGCGGGTAAAGATCGTGCGGGCTTCGGCACC 601 GCGCTGGTCCTGTCTGCTCTCGGTGTAGACAAAAACACCGTTATTGACGACTACATGCTG 661 TCTAACAAATACCGTGCGGACGAAAACAAGAAAGCTATCGAAGCGGTTGCGGCGAAAACG 721 GACAACAAAAAAGTTATCGACGGTATGACGGCGGTAATGGAAGTACGTGAATCTTACATT 781 AACGCGGCGTTCGACGAGATTAATGCGAAATACGGCTCTATGGACAACTTTCTGAAAGAA 841 AAGCTGGGTCTGACGGACGCGAAGAAAGAACAACTGAAGAAGGCGTATCTGTACTAA
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **


 * *GC% Content for gene (codon optimized): **51.5%

Do Not Need this info for Spring (but still copy these lines to your Target page for now) DNAWorks Results
 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): **

-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * ( link to DNA Works output text file - ** that should be saved in your Google Docs folder after you did the primer design protocol)

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

TATCCACCTTTACTGTTAGTACAGGTATGCTTTCT