Target-+Protein+Phosphatase+2B


 * Disease:** Trypansoma brucei - sleepy sickness
 * Enzyme:** [|Protein Phosphatase 2B]
 * Targe ** t: T. brucei TREU927 protein phosphatase 2B
 * NCBI Gene # ** : 71747666
 * Protein ID# **: XP_822888

Sleeping sickness, also known as African trypanosomiasis, is a vector-borne disease caused by the protozoa of the //Trypanosoma brucei// species. The tsetse fly is the insect that is responsible for transmitting the disease, and can only be found in the 36 s ub-Saharan Africa countries. There are two forms in which the //Trypanosoma brucei// species can cause sleeping sickness in humans: //Trypanosoma brucei gambiense and// //Trypanosoma brucei rhodesiense//. //Trypanosoma brucei gambiense// formaccounts for 98% of the reported sleeping sickness and located in West Africa while //Trypanosoma brucei rhodesiense// accounts for 2% of the reported sleeping sickness and is located in East Africa (1). The Eastern African form of sleeping sickness has shown to be more progressive than the Western African sleeping sickness (1, 2). Sleeping sickness is a potentially fatal disease that has become a public health care concern. It consists of two stages; the first has symptoms that include fever, headache and joint pain. The second stage is the neurological phase in which the parasite crosses the blood brain barrier and infects the central nervous system. This stage causes the disruption of the sleep cycle where sleep occurs during the daytime and wakefulness during nighttime (3). Other symptoms for this stage include tremor, confusion, hemiparesis, and general muscle weakness. Without treatment the disease is fatal (3, 4). Treatment in place today for this disease is pentamidine for stage one and melarsoprol, eflornithine, nifurtimox, and nifurtimox-eflornithine for stage two sleeping sickness (3). These treatments options are toxic however. In fact melarsoprol is the only drug that has been proven successful against both types of central nervous system disease, but is so toxic that it kills 5% of patients who receive it (5).Therefore there is a pressing need for a drug that will successfully treat both stages of this disease.
 * Background/Disease Information:**


 * pl:** 6.0436; **Amino Acid Length:** 431; **MW:** 48129 (Da); **Number of Paralogs:** 1
 * Essentiality:** Essential, used for [|cytokinesis]
 * Gene/Ortholog: ** Tb09.160.0480 (OG4_30044); ** Phenotype: ** significant loss of fitness in bloodstream forms (3 days); ** Source study: ** alsford
 * Metabolic Pathways:** gene is not mapped to metabolic pathways
 * Not complex**

**Drugability:** 0.8 (yes) **Link to Brenda:** http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
 * EC#:** 3.1.3.16

Query Coverage: 100% Max % Identities: 100%
 * BLAST:**


 * Current Inhibitors: ** no drug identifiers are listed


 * Expression Information (has it been expressed in bacterial cells): ** no expression information available
 * Purification Method: **

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/calcineurin.Par.0001.File.tmp/calcineurin.pdf http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
 * Assay:**


 * GC % Content for gene:** 47.4%


 * Reaction catalyzed by protein phosphatase 2B (from Brenda):**


 * Crystal Structure Image of Protein (PyMol):**

45350 - assuming all pairs of Cys residues form cystines 44350 - assuming all Cys residues are reduced
 * Molecular Weight in kiloDaltons using the [|Expasy ProtParam]website: ** 48128.6 Da
 * Molar Extinction coefficient at 280 nm wavelength: **


 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html).


 * Protein Sequence:**

MSSGASHHELTRGRDGLKEREYVWKKSHADESPGSNPQILPTLVLPHHLVFDNDGAPLAD

NIKVHFGRGWRLHVEDALNIVHRCALIMKEEPNVVRLKGSAVVCGDLHGQFHDLLTLLEV

NGHPSVQQYVFLGDYVDRGDFSAEIVLLCMSFKLLYPRSFILLRGNHESRQLTSCFNFKQ

EIESKYSSMVYEEIMAAFDCFPLSCVVNDRFFCVHGGLSPLLTYLGEIDTVNRFRETPST

GPMCDLLWSDPMFGDDTDCATPSEELFVFNTKRGCSYNYSYEAVCRFLEANNLCTVIRGH

ETQPGGYKLYRHTPKGVPAVVCVFSASNYCGTYGNMAAVVAIDGDVMNIRQYMATSHDSC

TPNHFNAISRMQPLAIHEAVEKWCVASHGGSSGDAKAEKVEVEKNLSEDDSSVVLREKLG

DMICAMHHIVT

Sequence Length: 431 aa

Coding Sequence:** GAATATGTTTGGAAAAAATCTCATGCAGATGAATCTCCCGGCAGTAATCCTCAGATACTT CCGACTCTCGTGTTGCCGCACCATTTGGTATTTGATAATGATGGCGCCCCGCTTGCAGAT AATATTAAAGTTCATTTTGGGAGAGGATGGCGACTTCATGTAGAGGATGCATTGAACATT GTCCACCGCTGTGCATTAATCATGAAAGAGGAGCCAAATGTTGTACGATTAAAGGGATCT GCTGTCGTGTGTGGGGATCTTCACGGGCAGTTTCATGACTTACTGACATTGTTAGAAGTT AATGGTCATCCCAGCGTGCAGCAATATGTTTTTCTTGGAGACTACGTGGACCGCGGGGAT TTTAGTGCAGAAATTGTACTCCTCTGCATGTCGTTTAAGCTACTGTACCCTCGTTCCTTC <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">ATACTACTACGTGGCAATCACGAATCACGACAACTAACGTCGTGTTTCAACTTCAAGCAG <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">GAGATTGAGAGCAAGTATTCGTCAATGGTGTACGAAGAGATTATGGCAGCGTTTGACTGC <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">TTTCCACTCTCATGTGTTGTAAATGATAGGTTTTTCTGTGTGCACGGCGGTTTATCGCCA <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">TTGTTAACTTATCTTGGAGAAATTGACACTGTTAACCGTTTCAGGGAAACACCGTCGACG <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">GGTCCCATGTGCGATTTGCTCTGGTCTGATCCCATGTTTGGAGACGACACCGACTGTGCA <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">ACTCCCTCTGAAGAGTTGTTTGTTTTCAATACCAAACGTGGTTGCTCATACAACTACAGT <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">TATGAGGCAGTTTGTCGGTTTCTGGAGGCGAATAATCTTTGCACAGTCATACGTGGCCAC <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">GAGACGCAGCCGGGAGGTTACAAACTGTATCGTCACACCCCGAAGGGAGTTCCAGCTGTG <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">GTTTGTGTTTTTTCCGCATCGAATTACTGTGGAACGTATGGAAATATGGCTGCCGTGGTA <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">GCAATTGATGGGGACGTGATGAATATTCGTCAATATATGGCGACCTCCCATGACTCCTGC <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">ACTCCAAATCATTTTAATGCAATTAGTCGTATGCAACCATTGGCAATACACGAGGCAGTT <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">GAAAAATGGTGTGTGGCTTCACACGGCGGAAGTAGTGGTGATGCAAAGGCAGAAAAGGTG <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">GAAGTAGAAAAAAATCTTTCGGAGGATGATTCTTCTGTGGTACTGCGGGAGAAGTTGGGG <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">GATATGATTTGTGCGATGCATCATATAGTTACTTGA
 * <span style="background-color: transparent; font-family: courier,fixed-width,monospace; font-size: 10pt; vertical-align: baseline;">ATGTCGAGTGGTGCTTCACACCATGAGCTAACACGTGGGCGTGATGGTTTGAAGGAACGG

Sequence Length: 1296 bp || atgagcctggatgcgctgaccaccctgccgattaaaaaacataccgcgctgctgaaccgc tttccggaaacccgctttgtgacccagctggcgaaaaaacgcgcgagctggattgtgttt ggccattatctgaccccggcgcagtttgaagatatggatttttttaccaaccgctttaac gcgattctggatatgtggaaagtgggccgctatgaagtggcgctgatggatggcgaactg accagcgaacatgaaaccattctgaaagcgctggaactggattatgcgcgcattcaggat gtgccggatctgaccaaaccgggcctgattgtgctggatatggatagcaccgcgattcag attgaatgcattgatgaaattgcgaaactggcgggcgtgggcgaagaagtggcggaagtg accgaacgcgcgatgcagggcgaactggattttgaacagagcctgcgcctgcgcgtgagc aaactgaaagatgcgccggaacagattctgagccaggtgcgcgaaaccctgccgctgatg ccggaactgccggaactggtggcgaccctgcatgcgtttggctggaaagtggcgattgcg agcggcggctttacctattttagcgattatctgaaagaacagctgagcctggattatgcg cagagcaacaccctggaaattgtgagcggcaaactgaccggccaggtgctgggcgaagtg gtgagcgcgcagaccaaagcggatattctgctgaccctggcgcagcagtatgatgtggaa attcataacaccgtggcggtgggcgatggcgcgaacgatctggtgatgatggcggcggcg ggcctgggcgtggcgtatcatgcgaaaccgaaagtggaagcgaaagcgcagaccgcggtg cgctttgcgggcctgggcggcgtggtgtgcattctgagcgcggcgctggtggcgcagcag aaactgagctggaaaagcaaagaaggccatcatcatcatcatcat
 * CDS Sequence:**

https://drive.google.com/?tab=wo&authuser=0#folders/0B4O2KqKh2q_-MlZNOWY4YWJJV0E
 * Link to DNA works output file:**

Forward Primer (33 base pairs) TACTTCCAATCC ATG TCTTCTGGCGCGTCCCAT (order) Melting Temperature 0 mM Mg 66.8 degC 1.5 mM Mg 73.6 degC 2 mM Mg 74.1 degC 4 mM Mg 75.0 degC 6 mM Mg 75.4 degC Reverse Primer (38 base pairs) GCGCAATGCACCACATTGTTACCCAGTAAAGGTGGATA Reverse complement TATCCACCTTTACTGGGTAACAATGTGGTGCATTGCGC (order) Melting Temperature 0 mM Mg 66.4 degC 1.5 mM Mg 73.7 degC 2 mM Mg 74.1 degC 4 mM Mg 74.9 degC 6 mM Mg 75.3 degC
 * Primer Design Results:**

Keely: Forward Primer(30bp)

TACTTCCAATCCATGTCTTCTGGCGCGTCC

Reverse Primer:

__<span style="font-family: courier,sans-serif; font-size: medium;">CACCACATTGTTACCTAA __CAGTAAAGGTGGATA

Reverse Complement(33bp)

TATCCACCTTTACTG__TTAGGTAACAATGTGGTG__

DNA Sequence from DNA Works with a TAA added to the end:

<span style="background-color: #ffffff; font-family: courier,sans-serif; font-size: medium;">__ATGTCTTCTGGCGCGTCC__CATCATGAGCTGACCCGTGGTCGTGATGGTCTCAAAGAACGT GAATACGTTTGGAAAAAATCTCACGCGGACGAGTCCCCGGGTTCTAACCCGCAAATCCTG CCGACCCTGGTTCTGCCGCACCACCTCGTGTTCGACAACGACGGTGCGCCACTGGCGGAC AACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCTCCACGTTGAGGATGCGCTGAATATC GTTCACCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCT GCAGTAGTCTGCGGTGATCTCCACGGCCAGTTCCACGACCTCCTGACCCTGCTGGAAGTT AATGGTCACCCTAGCGTTCAACAGTACGTTTTCCTGGGTGACTACGTTGACCGTGGTGAC TTCTCTGCTGAAATCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCCGCGCTCTTTC ATCCTGCTGCGTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAG GAAATCGAATCTAAATACTCTTCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGC TTCCCGCTGTCTTGCGTTGTTAACGACCGTTTCTTTTGTGTTCATGGTGGTCTCTCTCCG CTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGTGAAACCCCGTCTACT GGTCCGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACCGACTGTGCG ACGCCGAGCGAAGAACTGTTCGTTTTTAACACTAAACGTGGTTGCTCTTACAACTACAGC TACGAAGCCGTTTGCCGTTTCCTCGAAGCGAACAATCTGTGCACGGTTATTCGTGGTCAT GAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTGTTCCGGCGGTT GTATGTGTTTTTAGCGCGAGCAATTATTGCGGTACCTACGGTAACATGGCCGCAGTTGTA GCGATCGATGGTGACGTTATGAACATCCGTCAGTACATGGCGACCTCTCACGACTCTTGC ACCCCTAACCACTTCAATGCGATCTCTCGTATGCAGCCTCTGGCGATCCATGAGGCGGTA GAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAAAAAGTT GAGGTTGAAAAAAACCTGTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGC GATATGATCTGCGCAATG__CACCACATTGTTACCTAA__


 * < C2 ||< TBP2B 1 ||< ATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGAT ||
 * < C3 ||< TBP2B 2 ||< CCGCGTGAGATTTTTTCCAAACGTATTCACGTTCTTTGAGACCATCACGACCACGGGTCA ||
 * < C4 ||< TBP2B 3 ||< TTTGGAAAAAATCTCACGCGGACGAGTCCCCGGGTTCTAACCCGCAAATCCTGCCGACCC ||
 * < C5 ||< TBP2B 4 ||< GGCGCACCGTCGTTGTCGAACACGAGGTGGTGCGGCAGAACCAGGGTCGGCAGGATTTGC ||
 * < C6 ||< TBP2B 5 ||< CAACGACGGTGCGCCACTGGCGGACAACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCT ||
 * < C7 ||< TBP2B 6 ||< GATCAGCGCGCAACGGTGAACGATATTCAGCGCATCCTCAACGTGGAGGCGCCAACCGCG ||
 * < C8 ||< TBP2B 7 ||< CCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCTGCAGT ||
 * < C9 ||< TBP2B 8 ||< GTCAGGAGGTCGTGGAACTGGCCGTGGAGATCACCGCAGACTACTGCAGAGCCCTTCAGG ||
 * < C10 ||< TBP2B 9 ||< AGTTCCACGACCTCCTGACCCTGCTGGAAGTTAATGGTCACCCTAGCGTTCAACAGTACG ||
 * < C11 ||< TBP2B 10 ||< GCAGAGAAGTCACCACGGTCAACGTAGTCACCCAGGAAAACGTACTGTTGAACGCTAGGG ||
 * < C12 ||< TBP2B 11 ||< CCGTGGTGACTTCTCTGCTGAAATCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCC ||
 * < D1 ||< TBP2B 12 ||< CGAGACTCGTGGTTACCACGCAGCAGGATGAAAGAGCGCGGGTACAGCAGTTTGAAAGAC ||
 * < D2 ||< TBP2B 13 ||< GTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAGGAAATCGAAT ||
 * < D3 ||< TBP2B 14 ||< CCATGATTTCTTCGTAAACCATAGAAGAGTATTTAGATTCGATTTCCTGTTTGAAGTTGA ||
 * < D4 ||< TBP2B 15 ||< TCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGCTTCCCGCTGTCTTGCGTTGTT ||
 * < D5 ||< TBP2B 16 ||< CAGCGGAGAGAGACCACCATGAACACAAAAGAAACGGTCGTTAACAACGCAAGACAGCGG ||
 * < D6 ||< TBP2B 17 ||< GGTGGTCTCTCTCCGCTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGT ||
 * < D7 ||< TBP2B 18 ||< CAGAGCAGGTCACACATCGGACCAGTAGACGGGGTTTCACGAAAACGGTTAACAGTATCG ||
 * < D8 ||< TBP2B 19 ||< CGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACCGACTGTGCGACGC ||
 * < D9 ||< TBP2B 20 ||< AGAGCAACCACGTTTAGTGTTAAAAACGAACAGTTCTTCGCTCGGCGTCGCACAGTCGGT ||
 * < D10 ||< TBP2B 21 ||< AACACTAAACGTGGTTGCTCTTACAACTACAGCTACGAAGCCGTTTGCCGTTTCCTCGAA ||
 * < D11 ||< TBP2B 22 ||< TGGGTCTCATGACCACGAATAACCGTGCACAGATTGTTCGCTTCGAGGAAACGGCAAACG ||
 * < D12 ||< TBP2B 23 ||< TTCGTGGTCATGAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTG ||
 * < E1 ||< TBP2B 24 ||< CGCAATAATTGCTCGCGCTAAAAACACATACAACCGCCGGAACACCCTTTGGGGTATGGC ||
 * < E2 ||< TBP2B 25 ||< GCGCGAGCAATTATTGCGGTACCTACGGTAACATGGCCGCAGTTGTAGCGATCGATGGTG ||
 * < E3 ||< TBP2B 26 ||< TCGTGAGAGGTCGCCATGTACTGACGGATGTTCATAACGTCACCATCGATCGCTACAACT ||
 * < E4 ||< TBP2B 27 ||< CATGGCGACCTCTCACGACTCTTGCACCCCTAACCACTTCAATGCGATCTCTCGTATGCA ||
 * < E5 ||< TBP2B 28 ||< ACACACCACTTTTCTACCGCCTCATGGATCGCCAGAGGCTGCATACGAGAGATCGCATTG ||
 * < E6 ||< TBP2B 29 ||< GCGGTAGAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAA ||
 * < E7 ||< TBP2B 30 ||< CAGAGCTGTCATCTTCAGACAGGTTTTTTTCAACCTCAACTTTTTCCGCTTTCGCGTCAC ||
 * < E8 ||< TBP2B 31 ||< TGTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGCGATATGATCTGCGCAA ||
 * < E9 ||< TBP2B 32 ||< GGTAACAATGTGGTGCATTGCGCAGATCATATCGCC ||

References:
 * 1) Trypanosomiasis, Human African (sleeping sickness). //World Health Organization(WHO)// **2013.** []
 * 2) [|Parasites] - [|African Trypanosomiasis (also known as Sleeping Sickness)] . //Centers for Disease Control and Prevention (CDC)// **2012.** []
 * 3) Sleeping Sickness. //Doctors Without Borders// **2013.** []
 * 4) Brun, R.; Blum, J.; Chappuis, F.; Burri, C., Human African trypanosomiasis. //The Lancet// **2010**, 375, (9709), 148-159. []
 * 5) Kennedy, P. G.E, The continuing problem of human African trypanosomiasis (sleeping sickness). //Annals of Neurology// **2008,** 64, (2), 116-126. []