Target-serine-threonine+protein+phosphatase+(Leishmania+major)

Leishmania major, serine/threonine protein phosphatase, putative

Progress of Research:
LmajSTPP gene successfully constructed through Primary PCR and amplified by Secondary PCR.



Background and Introduction:

 * *Target (protein/gene name): ** serine/threonine protein phosphatase
 * *NCBI Gene # or RefSeq#: ** 5652543 (?)
 * *Protein ID (NP or XP #) or Wolbachia#: ** ?? (used Daniel's Oligo Design - Dr. B090313)

***Enzyme Assay information:** Assay for Calcineurin (3.1.3.16 ) ?? SIgma - just use PNPP via BRENDA ***Druggability:** 0.8 ***Essentiality Data** –yes *//Abundant Essentiality Data on TDR page//* []
 * *Organism (including strain): ** // Leishmania major //
 * *Etiologic Risk Group (see link below): ** Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents

[]

**__ EC#: __**3.1.3.16 []

**__ PDB # or closest PDB entry is using homology model __ : ** 3ICF (homology model- 94% query coverage) Comparison Protein BLAST Results:

Query Coverage: 94% Max % Identities: 39% % Positives 56% Chain used for homology: To be determined

KNAFKGAKIKNMSQEFISKMVNDLFLKGKYLPKKYVAAIISHADTLFRQEPSMVELENNSTPDVKISVCGDTHGQF YDVLNLFRKFGKVGPKHTYLFNGDFVDRGSWSCEVALLFYCLKILHPNNFFLNRGNHESDNMNKIYGFEDECKYK YSQRIFNMFAQSFESLPLATLINNDYLVMHGGLPSDPSATLSDFKNIDRFAQPPRDGAFMELLWADPQEANGMGP SQRGLGHAFGPDITDRFLRNNKLRKIFRSHELRMGGVQFEQKGKLMTVFSAPNYCDSQGNLGGVIHVVPGHGILQ AGRNDDQNLIIETFEAVEHPDIKPMAYSNGGFGL
 * >3ICF:A|PDBID|CHAIN|SEQUENCE **

<span style="color: black; font-family: Arial,Helvetica,sans-serif; size: 12px;">**__ Background/Disease __ : ** Leishmaniasis is a disease caused by protozoan parasites that belong to the genus of Leishmania and is transmitted by the bit of certain species of sand fly. <span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif;"> Leishmaniasis threatens about 350 million men, women and children in 88 countries around the world. As many as 12 million people are believed to be currently infected, with about 1–2 million estimated new cases occurring every year. <span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif; line-height: 1.5;">The disease can have a wide range of clinical symptoms, which may be **<span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif; line-height: 1.5; vertical-align: baseline;">cutaneous **<span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif; line-height: 1.5;">, **<span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif; line-height: 1.5; vertical-align: baseline;">mucocutaneous **<span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif; line-height: 1.5;"> or **<span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif; line-height: 1.5; vertical-align: baseline;">visceral **<span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif; line-height: 1.5;">. Cutaneous leishmaniasis is the most common form. Visceral leishmaniasis is the most severe form, in which vital organs of the body are affected.

(Extracellular dephosphorylation in the parasite, Leishmania major. This protein “may play a significant role in host cell-parasite recognition and infection.”)

<span style="background-color: #ffffff; color: #333333; font-family: Helvetica,Arial,sans-serif; line-height: 1.5;">__**Current Treatments:**__ There are two common therapies containing antimony (known as pentavalent antimonials ): meglumine antimoniate (Glucantime) and sodium stibogluconate (Pentostam). It is not completely understood how these drugs act against the parasite; they may disrupt its energy production or [|trypanothione] metabolism. Unfortunately, in many parts of the world, the parasite has become resistant to antimony when treating for visceral or mucocutaneous leishmaniasis, [|[5]] but the level of resistance varies according to species

Miltefosine (Impavido), is a new drug for visceral and cutaneous leishmaniasis. The cure rate of miltefosine in phase III <span style="background-color: #ffffff; color: #0b0080; font-family: sans-serif; line-height: 1.5; text-decoration: none;">[|clinical trials] is 95%; studies in Ethiopia show it is also effective in Africa. However, there are problems associated with the use of miltefosine that arise from its teratogenicity and pharmacokinetics: In a Dutch study by Thomas P.C. Dorlo in 2008, miltefosine was shown to be much slower eliminated from the body than previously thought and was therefore still detectable in human plasma samples taken five months after the end of treatment. The presence of subtherapeutic miltefosine concentrations in the blood beyond five months after treatment might contribute to the selection of resistant parasites and, moreover, the measures for preventing the teratogenic risks of miltefosine must be reconsidered. This led to some reluctance to taking Miltefosine by affected populations.

<span style="color: black; font-family: Arial,Helvetica,sans-serif; size: 12px;"> <span style="color: black; font-family: Arial,Helvetica,sans-serif; size: 12px;">**__PDB code:__** 3ICF
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 1.1em; line-height: 1.5;">__ Image of protein (PyMol or etc): __ **

<span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;">ATGACGACGGCCGGCGGCGGATCGGCGGTGGGGTCCAGCAGCGCCCTCGACTTGGATGAG <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> ATGATTAATTACGTCATACAGTGCAAGCCGCTCAGCGAACAGCAGGTCGCGCGCCTGTGC <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> GAGAAGGTGAAGGAGGTGCTGGAGAAGGAGAACAACGTGCACGCGGTGAGGGCGCCAGTC <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> ACGGTGTGCGGCGATGTGCATGGCCAGTTTCACGACTTGCTGGAGCTTTTCAAGATTGGC <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> GGGTTGCCGCCGGACACGAACTACCTGTTCATGGGGGACTACGTGGACCGCGGCTACTAC <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> AGCGTCGAGACGGTGACGCTGCTCCTCCTCTACAAGCTGCGATACCCCCAGCGACTTCAT <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> CTTCTCCGCGGCAACCACGAGTCGCGCCAGATCACGCAGGTGTATGGCTTCTACGATGAG <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> TGCATTCGCAAGTATGGTAGCGCCAACGTCTGGACGATCTTCACGGACCTGTTTGACTAC <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> CTGCCCCTGACAGCGTTGGTCGAGAACGACATCTTCTGCCTTCACGGTGGTCTCTCACCG <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> ACGGTCGACACATTCAGCCACATCCGCAACCTCGACCGCGTGCAGGAAGTGCCGCACGAG <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> GGGCCGATGTGCGATCTGCTATGGTCGGACCCGGACGATCGTGATGGCTGGGGCATCAGC <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> CCCCGTGGCGCCGGCTTCACCTTCGGCCAAGGAGTCACCGAAGGCTTCTGCCACAACAAC <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> AAGATCAAGACAATCGCCCGTGCGCATCAGCTTGTCATGGACGGCTACAGCTGGACGCAT <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> CAGGATCAGCTCGTCACGATCTTCAGCGCACCAAACTACTGCTACCGCTGCGGCAACCTC <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> GCCGGTCTTTTGGAGCTTGATGAGCACATGAACAAGTGCTTCTTTCAGTTCGACCCGGCG <span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 11pt; size: 12px;"> CCGCGACGCGGTGAAGCGCAGGTGTCGAAGAAGACGCCGGACTATTTTCTATAG
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 1.1em; line-height: 1.5;">__ CDS: __ **

<span style="color: black; font-family: Arial,Helvetica,sans-serif; size: 12px;">**__ Amino Acid Sequence: __**
XP_001683887.1

<span style="background-color: #ffffff; font-family: courier,fixed-width,monospace;">MTTAGGGSAVGSSSALDLDEMINYVIQCKPLSEQQVARLCEKVKEVLEKENNVHAVRAPV

<span style="background-color: #ffffff; font-family: courier,fixed-width,monospace;">TVCGDVHGQFHDLLELFKIGGLPPDTNYLFMGDYVDRGYYSVETVTLLLLYKLRYPQRLH

<span style="background-color: #ffffff; font-family: courier,fixed-width,monospace;">LLRGNHESRQITQVYGFYDECIRKYGSANVWTIFTDLFDYLPLTALVENDIFCLHGGLSP

<span style="background-color: #ffffff; font-family: courier,fixed-width,monospace;">TVDTFSHIRNLDRVQEVPHEGPMCDLLWSDPDDRDGWGISPRGAGFTFGQGVTEGFCHNN

<span style="background-color: #ffffff; font-family: courier,fixed-width,monospace;">KIKTIARAHQLVMDGYSWTHQDQLVTIFSAPNYCYRCGNLAGLLELDEHMNKCFFQFDPA

<span style="background-color: #ffffff; font-family: courier,fixed-width,monospace;">PRRGEAQVSKKTPDYFL

<span style="color: black; font-family: Arial,Helvetica,sans-serif; size: 12px;">**__ length of your protein in Amino Acids: __**
<span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 9pt; size: 12px;">317 aa

<span style="color: black; font-family: Arial,Helvetica,sans-serif; size: 12px;">**__ Molecular Weight of your protein __**
<span style="color: black; font-family: Arial,Helvetica,sans-serif; font-size: 10.5pt; size: 12px;">35861 Da

ATGACTACCGCTGGTGGTGGTTCTGCGGTGGGTTCTTCTTCCGCTCTCGACCTCGACGAA ATGATCAACTACGTTATCCAGTGCAAACCGCTGTCTGAACAGCAGGTTGCGCGCCTGTGC GAAAAAGTCAAAGAAGTTCTGGAGAAAGAAAACAACGTTCATGCGGTTCGTGCGCCGGTT ACCGTTTGCGGTGACGTTCACGGTCAATTCCACGACCTCCTGGAACTCTTCAAAATCGGT GGTCTGCCTCCGGACACCAATTACCTGTTCATGGGTGACTACGTCGATCGCGGTTATTAC AGCGTTGAAACGGTGACCCTCCTGCTGCTGTACAAACTGCGTTACCCGCAGCGTCTGCAC CTCCTCCGTGGTAACCACGAGTCTCGCCAGATTACTCAGGTTTACGGTTTCTACGACGAA TGCATCCGCAAATACGGCTCTGCGAATGTTTGGACCATCTTCACCGACCTCTTCGACTAC CTCCCGCTGACCGCGCTGGTTGAAAACGACATCTTCTGCCTGCACGGCGGTCTGTCTCCG ACCGTTGACACCTTCTCTCACATCCGTAACCTGGACCGTGTTCAGGAAGTTCCGCACGAG GGTCCGATGTGCGATCTGCTCTGGAGCGATCCGGACGACCGCGACGGTTGGGGTATCTCT CCGCGTGGTGCGGGTTTTACGTTCGGTCAGGGTGTAACCGAGGGTTTCTGCCACAACAAC AAAATCAAAACCATCGCGCGTGCGCATCAGCTGGTTATGGACGGTTACTCTTGGACCCAC CAAGATCAGCTCGTTACCATTTTCTCTGCGCCGAACTACTGCTACCGTTGCGGTAATCTC GCGGGTCTGCTGGAGCTGGACGAACACATGAATAAATGCTTTTTCCAATTTGACCCGGCG TAA
 * __Sequence of the gene produced from primer overlap:__**

__**Primers for pNIC-Bsa4 cloning:**__ Forward primers: TACTTCCAATCCATGACTACCGCTGGTGGTGG Reverse primers: TATCCACCTTTACTGTTACGCCGGGTCAAATTGG

Daniel's: Fin's: [|https://drive.google.com/?authuser=1#folders/0B4O2KqKh2q_-aTdxaGQzMWVUbXc] Manuel's:
 * 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)

__**References:**__ <span style="font-family: 'Calibri','sans-serif'; font-size: 14.6667px;">Homology Model Info <span style="font-family: 'Calibri','sans-serif'; font-size: 14.6667px;">SCRWL server [] []