Serine,+threonine+protein+phosphatase+2b+(catalytic+subunit),+putative+(P.+viva)

(use **Summer13D** Plate for Oligos instead of ordering new ones - Dr. B 090313)) 091213 - also use the **GDocs/Misc/Primers/DNAWorks_OutputSummer13** to get your correct DNA Works file for **Tail** Primer Design
 * Target (protein/gene name):** serine/threonine protein phosphatase 2b (catalytic subunit)
 * NCBI Gene # or RefSeq#:**156082205
 * Protein ID (NP or XP #) or Wolbachia#:** XP_001608591

To do: -Make homology model (Gdocs-Protocols-VirtualScreeningProtocols-HomologyModels-InstructionsProMolProject_HomologyModelversion6 (put in own folder) -Find 10-15 positive controls & 5 negative controls -Positive controls: binding database, ligands that stick to other similar organisms (PubChem) -ligand prep


 * Organism (including strain):** //Plasmodium vivax//
 * Etiologic Risk Group (see link below):** Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents

http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334 //Plasmodium vivax// is geographically the most widely distributed cause of malaria in people, with up to 2.5 billion people at risk and an estimated 80 million to 300 million clinical cases every year--including severe disease and death. Despite this large burden of disease, //P vivax// is overlooked and left in the shadow of the enormous problem caused by //Plasmodium falciparum// in sub-Saharan Africa. The technological advances enabling the sequencing of the //P vivax// genome and a recent call for worldwide malaria eradication have together placed new emphasis on the importance of addressing //P vivax// as a major public health problem. However, because of this parasite's biology, it is especially difficult to interrupt the transmission of //P vivax//, and experts agree that the available methods for preventing and treating infections with //P vivax// are inadequate. It is thus imperative that the development of new methods and strategies become a priority. Advancing the development of such methods needs renewed emphasis on understanding the biology, pathogenesis, and epidemiology of //P vivax//. This Review critically examines what is known about //P vivax//, focusing on identifying the crucial gaps that create obstacles to the elimination of this parasite in human populations.
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**


 * Essentiality of this protein: This protein is essential on the 6th day of development, where a knocked-out gene will cause //P. vivax// to loose fitness in bloodstream and procyclic forms.**
 * Complex of proteins?: No**
 * Druggable Target: Yes, Druggability index (0-1) of 0.8**
 * *EC#: 3.1.3.16 **

Spectrophotometric. Reagents: 200 mM Tris HCl Buffer, pH 7.5 at 30°C (Prepare 100 ml in deionized water, using Trizma Base, Sigma Prod. No. T-1503. Adjust to pH 7.5 at 30°C with 1 M HCl.) http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/calcineurin.pdf http://www.ncbi.nlm.nih.gov/pubmed/23233447 http://www.sigmaaldrich.com/catalog/product/sigma/c5207?lang=en®ion=US
 * Link to BRENDA EC# page:** http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
 * 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**

-- PDB # or closest PDB entry if using homology model: 2IE4_C -- For Homology Model option:
 * Structure Available (PDB or Homology model)**

Query Coverage: 72% Max % Identities: 55% % Positives 73% Chain used for homology: Chain C


 * Current Inhibitors:** Calcineurin was the only inhibitor found for serine/threonine protein phosphatase 2b (catalytic subunit), however, it was only studied in Humans, not //Plasmodium vivax.//
 * Expression Information (has it been expressed in bacterial cells):** Yes
 * Purification Method:** microcystin-Sepharose affinity chromatography

MEPLPNPKNDRQVKDVEPPPAKPLSLELLYPNGTDEPPDYKALRDHLKKEGRIRKEDCLDIIKRVIDIVS NEPNLLRLQDPITIVGDIHGQYYDFLKLLEVGGNPDNTQFLFLGDYVDRGSFSIEVLLLLYALKINFPHK IWLIRGNHECRQMTSFFNFRDECEYKYDMVVYYAFMESFDTIPLSAVINGKFLAVHGGLSPQLVLLNQIC SFTRFQEPPRSGIFCDILWADPIDEDKEEHTIQTESYFPNDIRGCSYFFGYNAATTFLEKNGLLSIIRAH EAQLEGYKMHQTNLKTGFPIVITIFSAPNYCDVYNNKGAVLKFDSNTLNIQQFSFSPHPYHLPNFMNLFT WSLPFVSEKVTEMLYCILNSSVNQSDEGVKDVVLPAEVLQIISYIEENNVKLEGMTLSGGGGATAGAAST GATEGSPSSQRKEALFKEGCFHSGASKEGGALGTTSPAAATATTQQMAAQGEQPAHLHTDDAQASKERSD ALRKKVQSVGRLMRVFRTLRKENELIVQLKGCSPGYRIPVGLLLQGKEGLENELEKFTKAKQIDSINEKR PPNE
 * Image of protein (PyMol with features delineated and shown separately):**
 * Amino Acid Sequence:**
 * length of your protein in Amino Acids:** 564
 * Molecular Weight:** 63 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 46925 M-1 cm-1
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html).



ATGGAGCCTCTACCAAACCCTAAGAACGATAGACAAGTGAAAGACGTGGAACCCCCACCAGCTAAGCCGC TGAGCCTGGAGCTGCTGTACCCGAACGGCACGGACGAGCCGCCAGACTACAAGGCGCTGCGCGACCACCT GAAGAAGGAGGGGCGCATCCGCAAGGAGGACTGCCTAGACATCATCAAGAGGGTGATCGACATCGTGAGC AACGAGCCAAACCTGCTGCGGCTCCAAGACCCAATAACGATCGTGGGGGACATCCACGGCCAGTACTACG ATTTTCTGAAGCTACTGGAAGTGGGAGGAAACCCAGATAACACTCAGTTCCTTTTCCTGGGCGACTACGT AGACAGAGGTTCCTTCAGTATAGAGGTCCTGCTCCTCCTCTACGCCCTCAAAATTAATTTCCCACATAAA ATATGGCTCATAAGAGGAAATCACGAGTGTAGGCAGATGACCTCCTTCTTCAATTTTAGAGACGAATGCG AATACAAATACGATATGGTTGTGTATTATGCCTTTATGGAATCCTTTGATACGATCCCCTTATCGGCTGT AATCAATGGGAAGTTCCTAGCCGTCCATGGGGGGTTGTCTCCTCAACTGGTACTCCTCAATCAGATATGT TCCTTCACGAGATTCCAAGAACCCCCACGGTCAGGGATTTTCTGTGACATTTTATGGGCGGACCCAATTG ACGAAGATAAAGAGGAGCATACCATTCAGACGGAATCCTACTTCCCCAATGACATCCGAGGGTGTAGCTA CTTCTTTGGCTATAATGCTGCCACCACCTTTTTAGAGAAGAACGGGTTACTCTCCATTATAAGAGCTCAT GAGGCACAGTTGGAGGGATACAAAATGCATCAAACCAATTTGAAGACCGGCTTCCCCATAGTCATCACCA TATTTTCTGCCCCTAATTATTGTGACGTTTATAATAACAAGGGGGCTGTACTCAAATTCGATAGCAACAC GTTGAACATCCAACAGTTTAGCTTTTCCCCCCACCCGTATCACCTCCCCAATTTTATGAACCTCTTCACC TGGTCTCTCCCCTTTGTTAGCGAGAAAGTCACAGAAATGCTCTACTGCATTTTAAACTCCAGCGTCAACC AATCGGATGAGGGGGTGAAGGACGTCGTGCTTCCTGCGGAGGTCCTCCAGATTATTAGCTACATAGAGGA GAACAATGTCAAGCTGGAGGGGATGACTCTAAGTGGTGGTGGTGGTGCTACTGCCGGTGCTGCTTCTACC GGTGCTACCGAGGGGTCCCCCTCCTCACAGAGGAAGGAAGCCCTTTTCAAGGAGGGCTGCTTCCACAGTG GGGCATCCAAAGAAGGGGGGGCATTGGGAACAACCTCCCCCGCTGCCGCTACTGCGACTACGCAGCAAAT GGCCGCCCAGGGGGAGCAACCGGCCCACCTGCACACCGATGACGCGCAGGCGTCCAAGGAACGGTCCGAC GCCCTCAGGAAAAAGGTTCAGTCAGTTGGTCGCCTGATGAGAGTGTTTAGGACGCTGCGCAAGGAGAACG AGTTGATTGTGCAGCTCAAGGGGTGCAGCCCCGGCTACCGCATCCCCGTGGGCCTCCTCCTGCAGGGCAA GGAGGGGCTGGAGAACGAGCTGGAGAAGTTCACCAAGGCCAAGCAGATCGACAGCATAAATGAGAAGCGG CCGCCGAACGAGTAG
 * CDS Gene Sequence:**


 * GC% Content for gene:** 52.3%

https://docs.google.com/file/d/0B4O2KqKh2q_-a1R3R1psN0ZWS0U/edit?usp=sharing
 * 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):**

http://www.ncbi.nlm.nih.gov/pubmed/19695492 http://www.ch.embnet.org/cgi-bin/TMPRED_form_parser http://www.tdrtargets.org/targets/view?gene_id=265333 http://www.ncbi.nlm.nih.gov/protein/156082205?report=fasta http://www.ncbi.nlm.nih.gov/nuccore/156082204?report=fasta http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/calcineurin.pdf
 * References:**

DVGGDIMNNDYIFLGDYVDRGYNSVETFEYLLLLKLLFPKNITLLRGNHESRQITTVYGF 127