Target+-+Quiescin+sulfhydryl+oxidase+-+Trypanosoma+brucei


 * Target (protein/gene name):** Quiescin sulfhydryl oxidase (QSOX)


 * NCBI Gene # or RefSeq#:** 88603894


 * Protein ID (NP or XP #) or Wolbachia#:** 845306


 * Organism (including strain):** Trypanosoma brucei


 * Etiologic Risk Group (see link below):** Risk Group 2


 * Background/ Disease Information  (sort of like the Intro to your Mini Research Write up):**

African sleeping sickness is a parasitic disease that can infect humans and other animals. It is caused by a protazoa of the species Trypanosoma brucei. These organisms are generally transmitted by the bite of the tsetse fly. It is most common in rural areas of Africa. The disease initially presents itself with symptoms including fever, headaches, itchiness, and joint pain. These symptoms generally begin 1 - 3 weeks after infection. Weeks to months later, the symptoms can progress to confusion, poor coordination, numbness, and trouble sleeping. African sleeping sickness is generally diagnosed via finding the parasite in a blood smear or in lymph fluid. A lumbar puncture can be used to determine whether a patient is in the first or second stage of the disease. Treatment of the disease is easier when it is in the first stage. Currently, the first stage is treated with the medications pentamidine or suramin. The second stage is treated with eflornithine or a combination of nifurtimox and eflornithine. If left untreated, African sleeping sickness usually, but not always, results in death. There are very few medically related prevention techniques for preventing the disease.


 * Link to TDR Targets page (if present):** N/A


 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)**

http://www.ncbi.nlm.nih.gov/protein/388603894


 * Essentiality of this protein:** QSOX is responsible for the formation of disulfide cross-links between cysteine side chains, which are important for protein stability, assembly, localization, and regulation.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521037/


 * Complex of proteins?:** Yes


 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):**

http://thorpelab.chem.udel.edu/research-program


 * *EC#: ** 1.8.3.2


 * Link to BRENDA EC# page:**
 * --** Show screenshot of BRENDA enzyme mechanism schematic


 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**

Sulfhydryl oxidase activity was measured at 25 °C by monitoring oxygen consumption in a Clarke-type oxygen electrode (Hansatech Instruments Ltd.). Buffer conditions were 50 mM potassium phosphate buffer, pH 7.5, 300 mM NaCl, 1 mM EDTA. HsQSOX1(PDI) and HsQSOX1(Erv) were mixed in the oxygen electrode chamber, and reactions were initiated by injection of DTT to a concentration of 1 mM. For comparison, //E. coli// thioredoxin was prepared as described and used in place of HsQSOX1(PDI). Intact HsQSOX1 activity was remeasured for reference by diluting the enzyme to 100 nM into the oxygen electrode chamber and initiating the reaction by injection of DTT to 1 mM. Turnover numbers were calculated per FAD cofactor. Oxygen consumption by HsQSOX1(Erv) alone is indistinguishable from background rates under these conditions (not shown). Oxygen consumption in the presence of varying concentrations of HsQSOX1(PDI) alone or thioredoxin alone is minimal.


 * -- link to Sigma (or other company  ) page for assay (see Sigma links below)**
 * -- -or link (or citation) to paper that contains assay information**

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521037/


 * --- List cost and quantity of substrate reagents, supplier, and catalog #**

(32048) Potassium phosphate buffer - $22.10 / L (S7653) NaCl - $33.90 / 250g (EDS) EDTA - $19.80 / 100g (D0632) DTT - $40.80 / 1g

-- PDB # or closest PDB entry if using homology model: 3QCP
 * Structure Available (PDB or Homology model)**

**Show pairwise alignment of your BLASTP search in NCBI against the PDB **

http://blast.ncbi.nlm.nih.gov/Blast.cgi

Max % Identities: 100%

% Positives: 100%


 * Current Inhibitors:** N/A


 * Expression Information (has it been expressed in bacterial cells):** Expressed in E. coli, BL21 (DE3) plysS strain


 * Purification Method :**

Cell pellets from 2 L of culture were resuspended in 20 mL of 50 mM potassium phosphate buffer, pH 7.5 containing 300 mM NaCl, 100 μM FAD and two tablets of protease inhibitor cocktail for His-tagged proteins (Complete, Mini, EDTA free; Roche). Cells were lysed by two passages through a French Press (at 10,000 psi) followed by brief sonication to shear DNA. The resulting lysate was centrifuged at 17,000 //g// for 30 min at 4 °C. The supernatant was carefully removed and added to Pro Bond Ni-NTA resin (Invitrogen) previously equilibrated in 50 mM phosphate buffer, pH 7.5 containing 300 mM NaCl. The suspension was rocked at 4 °C for 3 h and poured into an empty column. Flow-through was collected for further analysis and the column was washed with four column volumes of 50 mM phosphate buffer, pH 7.5 containing 300 mM NaCl followed by four column volumes of 50 mM phosphate buffer, pH 6.0. Protein bound to the resin was eluted using a gradient of imidazole in 50 mM phosphate buffer, pH 6.0 and collected in 1.5 mL aliquots. Fractions containing //Tb//QSOX enzyme were pooled and further purified using hydrophobic interaction chromatography. Here, ammonium sulfate was added to 40 % saturation at 4 °C and the solution clarified by centrifugation at 17,000 //g// for 30 min. The supernatant was then loaded onto a butyl-Sepharose column (6 × 2 cm) previously equilibrated with 50 mM phosphate buffer, pH 7.5 containing 40 % saturated ammonium sulfate and 1 mM EDTA. The column was washed with three column volumes of 50 mM phosphate buffer, pH 7.5, containing 40 % saturated ammonium sulfate and 1 mM EDTA. //Tb//QSOX was eluted with a decreasing gradient of ammonium sulfate. Fractions (5 mL) were pooled and concentrated using Amicon Ultra centrifugal filter devices (Millipore).


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




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

gshmsvaqva tgsprpglfh ldssvvdlsg ddfsrvhrva plcpwivlfy ndgcgacrry astfskfagg lkvehgkdal qiataaavnc asevdlcrky dinfvprlff fyprdscrsn eecgtssleh vafenshlev delesevrrl vnkhmvvdds lkercidmhf klytskeelv krsvsstdes grfvettely atdiagaffs amhydvslvg teprerltal edfvllvkds lpsigadgvv salesitaer pftvaswqda vvksgipfdg sprnvrwrtc rgsspqyrgf pcgmwlllha ltvntpadrn vleviqnyir yffsckecrd hfiqfnfspn edpvlqlwra hnnvnarlan vkdgadplvp krqfptleac tecydgagnf ieahvtgflk qrylwdpkav glmesnddln evdpaskdan vgrnvessgk gkgdggargn skevrsdhag


 * length of your protein in Amino Acids:** 470 aa


 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website:** 52.175 kDa


 * Molar Extinction coefficient  of your protein at 280 nm wavelength:** 50880


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