Target+-enoyl-acyl+carrier+reductase, Toxoplasma+gondii

Toxoplasma gondii is caused by the protozoan parasite. It is the most widespread parasitic infections and leads to congenital neurological birth dects. It is the third most common cause of food-borne deaths in the US. It infects 25% of the world population, usually through consumption of undercooked contaminate meat. No drugs to date have been made available to provide a fully effective treatment. Efforts have been made to interact with the enoyl reductase, which leadsto the inhibition of the fatty-acid biosynthesis pathway in the protozoan.
 * *Target (protein/gene name): **enoyl-acyl carrier reductase
 * *NCBI Gene # or RefSeq#: ** 7901395
 * *Protein ID (NP or XP #) or Wolbachia#: **AAQ74987.1
 * *Organism (including strain): ** Toxoplasma gondii
 * Etiologic Risk Group: ** Risk Group 2 Parasitic Agent
 * *Background/Disease Information: **


 * Essentiality of this protein: **The protein is essential in fatty-acid biosynthesis in the T. gondii. Without the enzyme, the protozoan lacks a crucial biomolecule.
 * Complex of proteins: **In complex with NAD and triclosan
 * Druggable Target: 0.8; **TCL ligand [triclosan]

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 * *EC#: ** 1.3.1.9
 * Link to BRENDA EC# page: **


 * -- ** Show screenshot of BRENDA enzyme mechanism

[] ) [] Substrate reagent: sulfadoxin [S7821] 10G = $64.50 25G = $158.50 http://www.sigmaaldrich.com/catalog/product/sigma/s7821?lang=en®ion=US&cm_sp=abstract-_-10779791-_-S7821
 * 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 **
 * Supplier: **Sigma-Aldrich

-- PDB # or closest PDB entry if using homology model: 2O2S -- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB
 * Structure Available (PDB or Homology model) **

 code Query 103  SAFPIDLRGQTAFVAGVADSHGYGWAIAKHLASAGARVALGTWPPVLGLFQKSLQSGRLD  162 SAFPIDLRGQTAFVAGVADSHGYGWAIAKHLASAGARVALGTWPPVLGLFQKSLQSGRLD Sbjct 1    SAFPIDLRGQTAFVAGVADSHGYGWAIAKHLASAGARVALGTWPPVLGLFQKSLQSGRLD  60

Query 163  EDRKLPDGSLIEFAGVYPLDAAFDKPEDVPQDIKDNKRYAGVDGYTIKEVAVKVKQDLGN  222 EDRKLPDGSLIEFAGVYPLDAAFDKPEDVPQDIKDNKRYAGVDGYTIKEVAVKVKQDLGN Sbjct 61   EDRKLPDGSLIEFAGVYPLDAAFDKPEDVPQDIKDNKRYAGVDGYTIKEVAVKVKQDLGN  120

Query 223  IDILVHSLANGPEVTKPLLETSRKGYLAASSNSAYSFVSLLQHFGPIMNEGGSAVTLSYL  282 IDILVHSLANGPEVTKPLLETSRKGYLAASSNSAYSFVSLLQHFGPIMNEGGSAVTLSYL Sbjct 121  IDILVHSLANGPEVTKPLLETSRKGYLAASSNSAYSFVSLLQHFGPIMNEGGSAVTLSYL  180

Query 283  AAERVVPGYGGGMSSAKAALESDTRTLAWEAGQKYGVRVNAISAGPLKSRAASAIGKSGE  342 AAERVVPGYGGGMSSAKAALESDTRTLAWEAGQKYGVRVNAISAGPLKSRAASAIGKSGE Sbjct 181  AAERVVPGYGGGMSSAKAALESDTRTLAWEAGQKYGVRVNAISAGPLKSRAASAIGKSGE  240

Query 343  KSFIDYAIDYSYNNAPLRRDLHSDDVGGAALFLLSPLARAVSGVTLYVDNGLHAMGQAVD  402 KSFIDYAIDYSYNNAPLRRDLHSDDVGGAALFLLSPLARAVSGVTLYVDNGLHAMGQAVD Sbjct 241  KSFIDYAIDYSYNNAPLRRDLHSDDVGGAALFLLSPLARAVSGVTLYVDNGLHAMGQAVD  300

Query 403  SRSMPPLQRATQEIN  417 SRSMPPLQRATQEIN Sbjct 301  SRSMPPLQRATQEIN  315 code 

Query Coverage: 75% Max % :100% % Positives: 100% Chain used for homology: 2O2S from PDB and NCBI protein AAQ74987.1


 * Current Inhibitors: ** antibacterial agent triclosan
 * Expression Information (has it been expressed in bacterial cells): ** Yes, it is expressed in BL21 E. Col, combined with the pMALc2x vector. Cells were grown in LB medium at 310 K to an optimal density.
 * Purification Method: **


 * Cells were resuspended in a lysis buffer. The buffer contains 20mM sodium/potassium phosphate, 20mM NaCl, and 2.5 micrograms of DNAse I.The lysate is **


 * centrifuged and applied onto a HiTrap Chelating HP column. TgENR (the protein) was eluted in a gradient and then desalted. The TgENR was then pooled and **

passed through a substractive amylose column to remove any residual fusion proteins, leaving us with pure TgENR fractions.


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

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

PDBID|CHAIN|SEQUENCE SAFPIDLRGQTAFVAGVADSHGYGWAIAKHLASAGARVALGTWPPVLGLFQKSLQSGRLDEDRKLPDGSLIEFAGVYPLD AAFDKPEDVPQDIKDNKRYAGVDGYTIKEVAVKVKQDLGNIDILVHSLANGPEVTKPLLETSRKGYLAASSNSAYSFVSL LQHFGPIMNEGGSAVTLSYLAAERVVPGYGGGMSSAKAALESDTRTLAWEAGQKYGVRVNAISAGPLKSRAASAIGKSGE KSFIDYAIDYSYNNAPLRRDLHSDDVGGAALFLLSPLARAVSGVTLYVDNGLHAMGQAVDSRSMPPLQRATQEIN code
 * *length of your protein in Amino Acids: ** 315
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: ** 2.008635797e+25 kDa

** Molar Extinction coefficient of your protein at 280 nm wavelength: ** 35870 M−1cm−1

 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

forward 5′-GGTGGT**GAATTC**TCAAACATAAACAAAATTAAAGAAG-3′ reverse 5′-GGTGGT**GTCGAC**TTATTCATTTTCATTGCGATATATATC-3′ Primers amplified nucleotides encoding amino-acid residues 103–417 of TgENR
 * *CDS Gene Sequence (paste as text only): **

http://www.brenda-enzymes.org/literature/lit.php4?e=1.3.1.9&r=671150


 * *GC% Content for gene: ** 50.3%


 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **
 * *GC% Content for gene (codon optimized): **

Don't need- -- 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 sequences 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)

**Resources:**
 * Primer design results for 'tail' primers (this is just 2 sequences): **

See ** ProtocolTargetDiscoveryVDS.docx ** for more Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334

@http://tdrtargets.org/
 * Databases of genes/organisms: **

@http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
 * Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations) **

NCBI GENE Page: @http://www.ncbi.nlm.nih.gov/gene BLAST Page: @http://blast.ncbi.nlm.nih.gov/
 * Gene Information: **

NCBI Protein Page: @http://www.ncbi.nlm.nih.gov/protein Protein Expression Website Protein Expression Paper:
 * Protein Information: **


 * Primer Overlap PCR Articles **

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