Target+-+S.+agalactiae+serine-threonine+phosphatase+STP1


 * To Do:**
 * make homology model
 * define active site using a similar enzyme (active site is probably the Mg ions - but align another structure

http://www.jbc.org/content/286/51/44197.long **http://www.ncbi.nlm.nih.gov/protein/YP_329040.1** A number of different bacteria, including //Escherichia coli// (//E.coli//), //Listeria//, and certain strains of streptococcus, may cause neonatal sepsis. Early-onset neonatal sepsis most often appears within 24 hours of birth. The baby gets the infection from the mother before or during delivery. hemolysin, autolysis, and virulence. Although not essential for growth, Stp1 is critical for GBS pathogenesis.
 * Target Discovery:**
 * Target (protein/gene name):** STP1, S. agalactiae serine/threonine phosphatase
 * NCBI Gene # or RefSeq#:** NCBI Gene ID 3686217
 * Protein ID (NP or XP #) or Wolbachia#:**YP_329040 (used Caroline's Oligo Design - Dr. B090313)
 * Get your NCBI NP# :**YP_329040
 * PDB #:** 2PK0/ UNITPROTKB: Q3K363
 * Organism:** Streptococcus agalactiae A909
 * Etiologic Risk Group:**
 * Background/Disease Information:** The disease is called neonatal septicemia or neonatal sepsis. It is a blood infection that occurs in infants younger than 90 days old.
 * Essentiality of this protein:** In Group B Streptococcus (GBS), Stp1 controls serine/threonine kinase function, post-transcriptional regulation of
 * Complex of enzymes**: No
 * EC#:** 3.1.3.16

http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.16
 * Link to BRENDA EC# page:**

Screenshot of BRENDA enzyme mechanism schematic:

http://www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en®ion=US http://www.brenda-enzymes.info/literature/lit.php4?e=3.1.3.16&r=666937
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):** reagents are phosphoprotein and water. It is spectrophotometric. **Not a couple assay.**
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates)**
 * -- link to paper that contains assay information**
 * -- List cost and quantity of substrate reagents and supplier:** Reagent is pNPP/ phosphoprotein. Cost on SIGMA $71.00. SKU-Packsize is P7998-100mL.
 * Structure Available (PDB or Homology model)**

Here's the PDB crystal structure of a yeast low molecular weight protein tyrosine phosphate complexed with the substrate PNPP.



Here's the PDB structure for the target protein, streptococcus agalactiae:



http://www.bindingdb.org/jsp/dbsearch/PrimarySearch_ki.jsp?target=Serine%2Fthreonine-protein+phosphatase&tag=tg&kiunit=nM&icunit=nM&column=ki&submit=Search&energyterm=kJ%2Fmole
 * -- PDB # or closest PDB entry if using homology model:** 2PKO - direct number but not for the A909 strand
 * -- Query Coverage (if not direct match):** 98%
 * -- Max Ident (if not direct match):** 495
 * Druggable Target (see Databases for this):** Yes
 * Current Inhibitors:**

from the Binding DB shows only 11 different molecules for my target.
 * Expression Information (has it been expressed in bacterial cells):** Escherichia coli bl21(de3)
 * Purification Method:** single-wavelength anomalous dispersion phasing and molecular replacement


 * Image of protein (PyMol or etc):**



MEISLLTDIGQRRSNNQDFINQFENKAGVPLIILADGMGGHRAGNIASEMTVTDLGSDWA ETDFSELSEIRDWMLVSIETENRKIYELGQSDDYKGMGTTIEAVAIVGDNIIFAHVGDSR IGIVRQGEYHLLTSDHSLVNELVKAGQLTEEEAASHPQKNIITQSIGQANPVEPDLGVHL LEEGDYLVVNSDGLTNMLSNADIATVLTQEKTLDDKNQDLITLANHRGGLDNITVALVYV ESEAV.
 * Amino Acid Sequence:**


 * Length of protein in Amino Acids:** 245
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website:** 26732.7
 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 18450

>gi|76786714:337442-338179 Streptococcus agalactiae A909 chromosome, complete genome ATGGAAATATCTCTTTTAACAGATATTGGGCAACGTCGTTCCAATAATCAAGATTTTATTAATCAATTTG AAAATAAAGCCGGCGTCCCACTAATCATCTTAGCAGATGGTATGGGAGGACATCGTGCAGGAAATATTGC TAGCGAGATGACTGTAACTGATCTAGGAAGTGATTGGGCGGAGACAGATTTTTCTGAGTTATCAGAAATT CGTGATTGGATGCTTGTCTCTATTGAAACAGAGAATCGCAAAATATATGAATTAGGTCAGTCAGATGATT ACAAAGGTATGGGAACTACCATTGAAGCAGTTGCCATTGTAGGAGATAATATTATTTTCGCACATGTAGG AGATTCACGTATCGGTATCGTTCGTCAAGGAGAGTATCATTTATTGACGAGTGATCATTCTCTTGTTAAT GAATTGGTAAAAGCTGGCCAATTAACAGAAGAAGAAGCTGCTAGTCATCCTCAAAAAAATATTATTACGC AATCAATTGGTCAAGCCAATCCAGTCGAACCTGATTTAGGAGTTCATCTACTTGAAGAAGGTGACTACCT TGTAGTTAATAGTGATGGACTAACTAATATGTTGAGTAATGCCGATATTGCTACAGTACTAACACAAGAA AAAACCTTAGATGATAAGAATCAAGATTTAATCACTCTAGCAAACCATAGAGGAGGTCTTGATAATATCA CTGTGGCACTAGTTTACGTTGAAAGTGAGGCGGTTTAA
 * CDS Gene Sequence:**


 * GC% Content for gene:** 37.127371273713 %

1 ATGGAAATCTCTCTCCTGACTGACATCGGTCAACGTCGCTCTAATAACCAGGACTTCATC  61 AATCAGTTCGAAAACAAGGCCGGTGTTCCGCTCATCATCCTGGCGGACGGCATGGGCGGT  121 CACCGTGCGGGTAACATTGCGAGCGAAATGACCGTTACCGATCTGGGCTCTGACTGGGCG  181 GAAACCGACTTCTCTGAACTGTCTGAAATCCGTGACTGGATGCTCGTTTCTATCGAAACG  241 GAAAACCGTAAAATCTACGAACTGGGTCAGTCTGACGACTACAAAGGTATGGGTACCACC  301 ATCGAAGCGGTTGCGATCGTTGGCGACAACATCATCTTCGCGCACGTTGGTGACTCTCGT  361 ATCGGTATCGTTCGTCAGGGTGAATACCATCTGCTGACTTCCGACCACTCTCTGGTTAAC  421 GAGCTGGTGAAAGCGGGTCAACTGACCGAAGAAGAAGCGGCGTCTCACCCGCAGAAGAAT  481 ATCATCACCCAGTCTATTGGCCAGGCGAACCCGGTTGAACCGGACCTGGGCGTCCACCTG  541 CTGGAAGAAGGTGACTACCTGGTTGTTAACTCTGACGGTCTGACCAACATGCTGTCTAAC  601 GCGGACATCGCGACCGTTCTGACGCAGGAAAAAACCCTGGACGACAAAAATCAGGACCTG  661 ATCACTCTCGCTAACCATCGTGGTGGTCTGGACAATATTACCGTTGCGCTGGTATACGTT  721 GAATCTGAAGCTGTTTAA
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol:**

52.30352303523%
 * GC% Content for gene (codon optimized):**

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

Brandy: https://drive.google.com/?tab=wo&authuser=0#folders/0B4O2KqKh2q_-VUNVTnM0UC1SYVU Caroline: https://drive.google.com/?tab=wo&authuser=0#folders/0B4O2KqKh2q_-Wk9IcXdqWmItRHM
 * Tail Primer Designs:** Entered 09/15/13 by Brandy


 * Primer design results for 'tail' primers (this is just 2 sequences): 100112 - These aren't tail primers -- Dr. B**
 * __ Upstream Primer __** : 5’ TACTTCCAATCC__ATGGAAATCTCTCTCCTG__ 3’ (<-- Whose information is this???) (Caroline)

GC Content: 43%

0 mM Mg2+ Tm:59.7˚C 1.5 mM Mg2+ Tm: 66.9˚C 2mM Mg2+ Tm: 67.4˚C

4mM Mg2+ Tm: 68.5˚C 6mM Mg2+ Tm: 69.0˚C


 * __ Downstream Primer __** : 5’ __GAATCTGAAGCTGTTTAA__CAGTAAAGGTGGATA 3’

GC Content: 36.4%

0 mM Mg2+ Tm:58.7˚C 1.5 mM Mg2+ Tm: 66.5˚C 2mM Mg2+ Tm: 67.0˚C

4mM Mg2+ Tm: 68.1˚C 6mM Mg2+ Tm: 68.6˚C


 * Forward and reverse primer design for insertion into pNIC-Bsa4 (Ruifei):** Uh Oh, You have 2 start codons in your forward! - Dr. B


 * Forward: TACTTCCAATCC __ ATGGAAATCTCTCTCCTC __**
 * Reverse: TATCCACCTTTACTG <span style="background-color: #ff0000; color: #000000; font-family: Arial; font-size: 15px; vertical-align: baseline;">__TTA__ <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; vertical-align: baseline;">__TTCTTCGTCCGTCAC__ **


 * Table 1: GC content and melting temperature as a function of the concentration of Mg++ for **CORRECTED** forward and reverse primers for insertion of S. agalactiae serine/threonine phosphatase gene into pNIC-Bsa4 vector with 0.4 uM Oligo concentration, 50 mM Na+ concentration, 0.3 mM dNTP concentration


 * || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">GC Content || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">T(m) at 0 mM Mg++ (°C) || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">T(m) at 1.5 mM Mg++ (°C) || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">T(m) at 2 mM Mg++ (°C) || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">T(m) at 4 mM Mg++ (°C) || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">T(m) at 6 mM Mg++ (°C) ||
 * <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">Forward primer || 43.3 || 59.5 || 66.7 || 67.2 || 68.3 || 68.8 ||
 * <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">Reverse Primer || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">42.4% || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">61.0 || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">68.3 || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">68.8 || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">69.8 || <span style="background-color: transparent; font-family: Arial; font-size: 15px; vertical-align: baseline;">70.3 ||

TMpred graph:



**Resources:**


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">GBS Phosphatase: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.jbc.org/content/286/51/44197.long__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Proof that A909 causes sepsis: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.straininfo.net/genomes/326__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">PDB: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.rcsb.org/pdb/explore/explore.do?structureId=2PK0__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">NCBI: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.ncbi.nlm.nih.gov/protein/AAL58473.1__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Pubmed on neonata sepsis: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0004557/__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">ExPASy: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://enzyme.expasy.org/cgi-bin/enzyme/enzyme-search-ec__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">SIGMA for PnPP: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en&region=US__] **


 * <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.sigmaaldrich.com/technical-documents/articles/biofiles/colorimetric-alkaline.html__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">SIGMA FOR ENZYME ASSAY: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/calcineurin.Par.0001.File.tmp/calcineurin.pdf__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">Price of pNPP on SIGMA: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en&region=US__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">PDB structure of pNPP with phosphotase, etc: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.rcsb.org/pdb/explore/explore.do?structureId=1D1Q__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">PDB Structure for STP 1: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://www.rcsb.org/pdb/explore/images.do?structureId=2PK0__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">COMBINED PROTEIN RESULTS: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://web.expasy.org/cgi-bin/protparam/protparam__] **


 * <span style="background-color: transparent; color: #000000; font-family: Arial; font-size: 15px; text-decoration: none; vertical-align: baseline;">ARticle used for purification: <span style="background-color: transparent; color: #1155cc; font-family: Arial; font-size: 15px; vertical-align: baseline;">[|__http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05845.x/full__] **