Target-cytosolic+malate+dehydrogenase+(Trypanosoma+brucei)

http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334 Trypanosoma brucei is the protozoa that is the cause for the parasitic disease African trypanomiasis. This disease can infect both humans and animals, and is characterized by fever and headaches, until the lymphatic and circulatory systems become affected. The disease then progresses rapidly and has a variety of manifestations including severely disrupted sleep cycles due to the parasite passing through the blood brain barrier, anemia, endocrine malfunctions, cardiac problems and kidney dysfunctions. However, the presence of orthologues of this gene in both //T. cruzi // and //L. major // suggests that it must fulfill some important function in trypanosomatids. http://www.sciencedirect.com/science/article/pii/S0020751905003140 [] [] [] 7.3. REAGENTS 7.3.1. 100mM Potassium Phosphate Buffer, pH 7.5 at 25ºC (Buffer)  $ 19.90 Prepare a 13.6 mg/mL solution in purified water using Potassium Phosphate such as [|Sigma-Aldrich Product Number P5379]. Adjust to pH 7.5 at 25°C with 1.0 N KOH. 7.3.2. 0.14mM β-Nicotinamide Adenine Dinucleotide Solution (β-NADH)  $23 Prepare a 0.14mM solution, corrected for water content, in Reagent 7.3.1 (Buffer), using β-Nicotinamide Adenine Dinucleotide Solution (reduced form), such as [|Sigma-Aldrich Product Number N8129]. Prepare Fresh. 7.3.3. 7.6mM Oxalacetic Acid (OAA)  $35.80 Immediately prior to use, prepare a 1.0 mg/mL solution in Reagent 7.3.1 (Buffer), using Oxalacetic Acid, such as [|Sigma-Aldrich Product Number O4126]. Product is not stable once in solution. Prepare Fresh For Each Kinetics Run. 7.3.4. Malic Dehydrogenase (MDH)  $25 Immediately before use, prepare a 0.2-0.5 units/mL solution in cold Reagen 7.3.1 (Buffer). Prepare Fresh.
 * Target (protein/gene name):** Cytosolic malate dehydrogenase
 * NCBI Gene # or RefSeq#:**15077032
 * Protein ID (NP or XP #) or Wolbachia#:** AAK83037.1
 * Organism (including strain):** Trypanosoma brucei
 * Etiologic Risk Group (see link below):** Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**
 * Essentiality of this protein: **
 * Complex of proteins?:** No
 * Druggable Target:** 0.8
 * *EC#: 1.1.1.37 **
 * Link to BRENDA EC# page:**
 * 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 **

-- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB ignment statistics for matc Query Coverage: 92% Max % Identities: 51% % Positives: 64% Chain used for homology: 4MDH (Chain A)  Sequence ID: lcl|60447  Length: 328 Number of Matches: 1   <span style="background-color: #ffffff; color: #222222; display: block; font-family: Verdana,sans-serif; font-size: 11px;"> Related Information  <span style="background-color: #ffffff; color: #222222; display: block; font-family: arial,sans-serif;"> <span style="display: block; font-family: Verdana,sans-serif; font-size: 11px;"> Range 1: 7 to 311  60447&tracks=[key:sequence_track,name:Sequence,display_name:Sequence,id:STD1,category:Sequence,annots:Sequence,ShowLabel:true][key:gene_model_track,CDSProductFeats:false][key:alignment_track,name:other%20alignments,annots:NG%20Alignments|Refseq%20Alignments|Gnomon%20Alignments|Unnamed,shown:false]&v=0:326&appname=ncbiblast&link_loc=fromHSP|Graphics  Alignment statistics for match #1 ||~ Score code Query 5    RVLVTGAAGQIAYSLLYSIGNGSVFGKDQPIILVLLDITPMMGVLDGVLMELQDCALPLL  64 RV VTGAAGQI YSLL I  G + G DQ + L LLDI+P +  L+G+  EL DC+ PLL Sbjct 7    RVAVTGAAGQIGYSLLPLIAAGRMLGFDQRVQLQLLDISPALKALEGIRAELMDCSFPLL  66
 * Structure Available (PDB or Homology model)**
 * ~ Expect ||~ Method ||~ Identities ||~ Positives ||~ Gaps ||
 * 305 bits(780) || 8e-106 || Compositional matrix adjust. || 158/309(51%) || 199/309(64%) || 5/309(1%) ||

Query 65   KDVIATDKEEIAFKDLDVAILVGSMPRRDGMERKDLLKANVKIFKCQGAALDKYAKKSVK  124 V+ TD+ ++AF  D+AIL G+ PR+ GMER+DLL+ N KIF  QG  L + A  + + Sbjct 67   DGVVITDEPKVAFDKADIAILCGAFPRKPGMERRDLLQTNAKIFSEQGRVLGEVASPNCR  126

Query 125  VIVVGNPANTNCLTASKSAPSIPKENF-SCLTRLDHNRAKAQIALKLGVTSDDVKNVIIW  183 V VVGNPANTN L  + +       F + LTRLDHNRA AQ+A +     ++VKN IIW Sbjct 127  VCVVGNPANTNALILLRESKGKLNPRFVTALTRLDHNRATAQVAERARARVEEVKNCIIW  186

Query 184  GNHSSTQYPDVNHAKVKLQAKEVGVYEAVKDDSWLKGEFITTVQQRGAAVIKARKLSSAM  243 GNHS TQ PDVN A V +        AV +D++   EFIT VQ+RGA ++K R LSSA+ Sbjct 187  GNHSGTQVPDVNSATVGGKPARAAVDNDAFFDNEFITIVQERGAEIMKLRGLSSAL  242

Query 244  SAAKAICDHVRDIWFGTPEGEFVSMGIISDGNSYGVPDDLLYSFPVTIKDKTWKIVEGLP  303 SAAKAI DHV D  GTP G  VSM + SDGN YGVP  L++SFPVT     W+IV GL Sbjct  243  SAAKAIVDHVHDWMLGTPSGTHVSMAVYSDGNPYGVPGGLIFSFPVTCSGGEWQIVSGLN  302

Query 304  INDFSREKM  312 +    E++ Sbjct 303  VTPAISERI  311

code
 * Current Inhibitors: ** 7 hits were found in the binding database, the top hit being CHEMBL318778.

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 * Expression Information (has it been expressed in bacterial cells): ** The protein is usually expressed via its natural protozoa, but has been cloned and characterized from e.coli before, <span style="background-color: #ffffff; color: #2e2e2e; font-family: 'Arial Unicode MS','Arial Unicode',Arial,'URW Gothic L',Helvetica,Tahoma,sans-serif; line-height: 1.5; text-align: justify;">pET28(a <span style="color: #2e2e2e; font-family: 'Arial Unicode MS','Arial Unicode',Arial,'URW Gothic L',Helvetica,Tahoma,sans-serif; font-size: 0.75em; line-height: 1.5; text-align: justify; vertical-align: super;">+ <span style="color: #2e2e2e; font-family: 'Arial Unicode MS','Arial Unicode',Arial,'URW Gothic L',Helvetica,Tahoma,sans-serif; line-height: 1.5; text-align: justify;">) and pET24(a <span style="color: #2e2e2e; font-family: 'Arial Unicode MS','Arial Unicode',Arial,'URW Gothic L',Helvetica,Tahoma,sans-serif; font-size: 0.75em; line-height: 1.5; text-align: justify; vertical-align: super;">+ <span style="color: #2e2e2e; font-family: 'Arial Unicode MS','Arial Unicode',Arial,'URW Gothic L',Helvetica,Tahoma,sans-serif; line-height: 1.5; text-align: justify;">) were used as expression vectors in bacteria.
 * Purification Method: ** A<span style="background-color: #ffffff; color: #2e2e2e; font-family: 'Arial Unicode MS','Arial Unicode',Arial,'URW Gothic L',Helvetica,Tahoma,sans-serif; line-height: 1.5; text-align: justify;">garose gel purification the PCR products were cloned into pGEM-T-easy vector and fully sequenced

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 * Image of protein (PyMol with features delineated and shown separately):**

<span style="background-color: #ffffff; font-family: courier,sans-serif; font-size: medium;">msntckrvavtgaagqigysllpliaagrmlgfdqrvqlqlldispalkalegiraelmd csfplldgvvitdepkvafdkadiailcgafprkpgmerrdllqtnakifseqgrvlgev aspncrvcvvgnpantnalillreskgklnprfvtaltrldhnrataqvaerararveev knciiwgnhsgtqvpdvnsatvggkparaavdndaffdnefitivqergaeimklrglss alsaakaivdhvhdwmlgtpsgthvsmavysdgnpygvpgglifsfpvtcsggewqivsg <span style="background-color: #ffffff; font-family: courier,sans-serif; font-size: medium;">lnvtpaiserikattteleeerrevsta
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**
 * length of your protein in Amino Acids:** 328

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 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website** 35209.3
 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 21345


 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * CDS Gene Sequence (paste as text only):**
 * GC% Content for gene:**
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**
 * GC% Content for gene (codon optimized):**