Dihydrofolate+reductase+-+thymidylate+synthase+(Trypanosoma+brucei)


 * ** Target (protein/gene name): **Dihydrofolate reductase – thymidylate synthase
 * ** NCBI Gene # or RefSeq#: **3658761
 * ** Protein ID (NP or XP #) or Wolbachia#: ** XP_846171.1
 * ** Organism (including strain): **//Trypanosoma brucei //TREU927
 * ** Etiologic Risk Group (see link below): ** Risk Group 2
 * **Background/Disease Information** (sort of like the Intro to your Mini Research Write up):
 * The protozoan organism Trypanosoma brucei is responsible for the disease African Trypanosomiasis, also known as African Sleeping Sickness. The organism is considered a Risk Group 2 organism and is considered a neglected tropical disease, according to TDR Targets Database. The protist is transmitted through the bite of a tsetse fly and it is actually possible for a person to be infected with this disease yet show no sign of any symptoms until many months or years later. Without any treatment, this disease occurs in two stages: the first stage symptoms include fever, joint pain and itching and in the second stage includes infection to the central nervous system, causing changes in behavior, loss of certain motor functions and coordination. The damage to the central nervous system can affect normal sleep patterns and thus gives its name. There is currently no known drug to treat African Trypanosomiasis.
 * ** Essentiality of this protein: ** After six days, this protein is essential to the organism.
 * 1) **Gene/Ortholog:** Tb927.7.5480 (OG4_10927); **Phenotype:** no significant loss or gain of fitness in bloodstream forms (3 days); **Source study:** alsford
 * 2) **Gene/Ortholog:** Tb927.7.5480 (OG4_10927); **Phenotype:** significant loss of fitness in bloodstream forms (6 days); **Source study:** alsford
 * 3) **Gene/Ortholog:** Tb927.7.5480 (OG4_10927); **Phenotype:** significant gain of fitness in procyclic forms; **Source study:** alsford
 * 4) **Gene/Ortholog:** Tb927.7.5480 (OG4_10927); **Phenotype:** significant gain of fitness in differentiation of procyclic to bloodstream forms; **Source study:**alsford
 * **alsford: ** High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 2011, 21:915-924
 * ** Complex of proteins?: ** Yes
 * ** Druggable Target: ** 1
 * ** EC#: ** 1.5.1.3
 * ** Link to BRENDA EC# page: **http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.5.1.3
 * http://www.brenda-enzymes.org/literature/lit.php4?e=1.5.1.3&r=392285
 * Link to a PubMed article that studied DHFR-TS gene isolation and expression

>>> >>> B. 0.11 mM ß -Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form Solution >>> ( ß -NADPH) ( Sigma Stock No. N9660) >>> Pricing is for NADPH, reduced tetra (cyclohezylammonium) salt Sigma Stock No. N5130:$99.90 for 25mg >>> >>> C. 2.3 mM Dihydrofolic Acid Solution (DHFA) (Sigma Prod. No. D-7006) >>> <span style="color: red; font-family: Arial,sans-serif; font-size: 10pt; line-height: 1.5;">Price: $71.60 for 10mg >>> >>> <span style="font-family: Arial,sans-serif; font-size: 10pt;">D. 0.1% (w/v) Bovine Serum Albumin Solution (BSA) >>> (Sigma Prod. No. A-4503) <span style="color: red; font-family: Arial,sans-serif; font-size: 10pt;">Price: $73.60 for 10g >>> >>> <span style="font-family: Arial,sans-serif; font-size: 10pt;">E. Dihydrofolate Reductase Enzyme Solution >>> <span style="color: red; font-family: Arial,sans-serif; font-size: 10pt; line-height: 1.5;">Dihydrofolate Reductase (Human) Sigma No. D6566: $241.50 for .25 units >>>
 * ** Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **
 * link to Sigma: <span style="font-family: Calibri,sans-serif; font-size: 11pt;">http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/dihydrofolatereductase.pdf
 * link (or citation) to paper that contains assay information: <span style="font-family: Calibri,sans-serif; font-size: 11pt;">http://www.ncbi.nlm.nih.gov/pubmed/11325838
 * ** List cost and quantity of substrate reagents and supplier **
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">A. 50 mM Potassium Phosphate Buffer, pH 6.5 at 25 ° <span style="font-family: Arial,sans-serif; font-size: 10pt;">C (Sigma Prod. No. P-5379) <span style="color: red; font-family: Arial,sans-serif; font-size: 10pt;">$19.90 for 100g
 * ** Structure Available (PDB or Homology model) **
 * PDB #: 3RG9

> <span style="font-family: Calibri,sans-serif; font-size: 11pt;">[] > <span style="font-family: Calibri,sans-serif; font-size: 11pt;">[]
 * ** Current Inhibitors: **<span style="font-family: Calibri,sans-serif; font-size: 11pt; line-height: 1.5;">7,8-dihydrofolate + NADPH
 * Expression Information (has it been expressed in bacterial cells): **<span style="font-family: Calibri,sans-serif;">Information is shown in the experimental procedure section in link below. **
 * Purification Method: **<span style="font-family: Calibri,sans-serif;">Information is shown in the experimental procedure section in link below. **
 * ** Image of protein (PyMol with features delineated and shown separately): **




 * ** Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **

MLSLTRILRKKIPVHELAGKISRPPLRPFSVVVASDEKGGIGDGGTIPWEIPEDMQYFRR VTTNLRGKNVKPSPSKRNAVVMGRKTWDSLPPKFRPLSNRLNVVLSRSATKEQLLAGIPD PIKRAEAANDVVAVNGGLEDALRMLVSKEHTSSIETVFCIGGGTIYKQALCAPCVNVLQA IHRTVVRPASNSCSVFFDIPAAGTKTPEGLELVRESITDERVSTGAGGKKYQFEKLVPRN SEEEQYLNLVGRIIDEGCTKCDRTGVGTRSLFGAQMRFSLRNNRLPLLTTKRVFWRGVCE ELLWFLRGETNAKLLSDKGIHIWDGNGSRAFLDSRGLTDYDEMDLGPVYGFQWRHFGADY ISCKVDSEGKGVDQIANIVKSLIENPDDRRMICTAWNPAALPRMALPPCHMMAQFYVSNG ELSCMLYQRSCDMGLGVPFNIASYALLTFLMAKASGLRPGELVHTLGDAHVYSNHVEPCR KQLKRVPRXFPFIVFKQDKEFLEDFQESDIEVIDYSPYPVISMEMAV


 * ** Length of your protein in Amino Acids: ** 527
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website: 58.8 kDa
 * ** Molar Extinction coefficient of your protein at 280 nm wavelength: ** ** 58620 ** M -1 cm -1
 * ** TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.



TMpred indicates that it is possible that there are two transmembrane helices.

> TATCCCTGATCCCATCAAACGAGCGGAGGCTGCGAATGATGTGGTTGCTGTGAACGGTGGGCTTGAAGAC > GCACTTCGGATGTTGGTAAGTAAGGAGCACACATCTTCTATCGAAACGGTGTTTTGCATTGGTGGCGGGA > CTATATATAAACAAGCCCTTTGTGCCCCATGCGTTAATGTGTTGCAAGCTATCCACCGGACGGTGGTCCG > ACCAGCCTCAAATTCGTGTAGCGTTTTCTTTGACATCCCAGCCGCAGGGACAAAGACCCTGGAAGGACTT > GAGTTGGTGCGGGAAAGCATAACGGACGAACGGGTATCAACCGGAGCTGGCGGAAAGAAATATCAGTTTG > AGAAGCTTGTCCCCCGAAACAGCGAAGAGGAGCAGTATTTAAATCTCGTTGGCCGCATAATTGATGAGGG > ATGTACGAAATGTGACCGCACGGGTGTTGGTACCCGCTCCCTCTTTGGGGCCCAGATGCGTTTCTCTCTC > CGCAACAATCGCCTGCCATTACTAACCACGAAGCGTGTCTTTTGGCGTGGAGTATGTGAAGAGCTGTTGT > GGTTTCTTCGGGGAGAAACAAATGCAAAGTTGCTGAGCGACAAGGGGATTCACATATGGGATGGGAATGG > CTCACGAGCATTCCTTGACAGTCGTGGCTTGACTGATTATGATGAGATGGACCTCGGACCTGTTTATGGC > TTCCAGTGGAGGCATTTTGGTGCCGACTATATTTCTTGCAAGGAGGATTCCGAGGGGAAAGGTGTTGATC > AGATTGCAAACATTGTGAAGTCATTGATAGAGAACCCGGATGACCGTCGCATGATTTGTACAGCTTGGAA > TCCGGCCGCCTTGCCACGCATGGCGCTCCCACCATGCCACATGATGGCGCAATTCTACGTGAGCAATGGG > GAACTCTCATGCATGTTGTACCAGCGGTCATGTGATATGGGTCTTGGCGTCCCCTTCAATATTGCTTCGT > ATGCACTGCTTACATTCCTCATGGCAAAAGCTTCTGGACTTCGGCCGGGCGAATTGGTGCACACCCTTGG > CGATGCACATGTTTACAGTAACCATGTGGAGCCGTGTCGGAAGCAATTGAAACGCGTGCCCCGCCCGTTC > CCTTTCATCGTGTTCAAACAAGACAAAGAGTTCCTGGAAGACTTTCAGGAAAGCGATATAGAGGTCATCG > ATTATTCTCCGTACCCCGTAATTTCTATGGAGATGGCGGTGTAG
 * CDS Gene Sequence (paste as text only): TCCGTCCACTGTCTAACCGCCTTAATGTTGTGCTGTCACGAAGTGCGACAAAGGAACAGTTGTTGGCTGG
 * GC% Content for gene: <span style="font-family: Calibri,sans-serif; font-size: 11pt; line-height: 1.5;">50.88%
 * ** CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **
 * ** GC% Content for gene (codon optimized): **

Do Not Need this info for Spring (but still copy these lines to your Target page for now) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * 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 TDR Targets Page: http://tdrtargets.org/targets/view?gene_id=13461 Pubmed - T. cruzi DHFR-TS: http://www.sciencedirect.com/science/article/pii/0166685194900760 Pubmed - T. brucei DHFR-TS: http://www.sciencedirect.com/science/article/pii/016668519500059A Pubmed - T. cruzi DHFR-TS: http://www.sciencedirect.com/science/article/pii/S096808961000324X