TargetSp15+-+Thioredoxin+Reductase+(Mycobacterium+tuberculosis)

Thioredoxin reductase/trxB2
 * *Target (protein/gene name): **

886232
 * *NCBI Gene # or RefSeq#: **

NP_218430.1
 * *Protein ID (NP or XP #) or Wolbachia#: **

//Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) //
 * *Organism (including strain): **

Risk group 4 (high individual risk but low community risk)
 * Etiologic Risk Group: **

Pulmonary tuberculosis is caused by //Mycobacterium// //tuberuculosis.// Tuberculosis is highly infectious with just droplets ejected from an infected person (cough, sneezes) able to grow //M. tuberculosis// in the lungs of another person. Though infectious, pulmonary tuberculosis is likely not to develop in a person with a strong immune system. However, due to poor healthcare in many developing and undeveloped countries, tuberculosis is the second most common infectious disease in the world. Macrophages phagocytose //M. tuberculosis// in the alveoli, where people with healthy immune systems successfully contain tuberculosis. However, in weak immune systems, white blood cells are not as efficient in stopping/slowing the growth of //M. tuberculosis//. Symptoms of tuberculosis include but is not limtied to coughing, chest pain, fatigue, and fever. Treatment for tuberculosis often takes between 6-9 months with mulitple drugs. There are many theories of why tuberculosis is difficult to treat; One possible reason is the observed asymmetrical division of //M. tuberculosis//, which leads to cells of varying growth rates, drug resistance, and development stage. It has been discovered that cells early in development are more susceptible to rifampin, and cell later in development is more susceptible to isoniazid and cycloserine. Traditional drug cocktails to treat tuberculosis may contain isoniazid, rifampin, ethambutol, and pyrazinamide (list is not exhaustive). New drugs are being developed due to developed drug resistance.
 * */ Disease Information (sort of like the Intro to your Mini __Research Write__ up): **

n/a
 * Link to TDR Targets page (if present): **

**NCBI**: http://www.ncbi.nlm.nih.gov/gene/886232#general-protein-info
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) **

Txb2 is essential to M. tuberculosis due to being a major disulide reductase and functions in pyrimidine conversions.
 * Essentiality of this protein: **

Is it a monomer or multimer as biological unit ** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer

No
 * Complex of proteins?: **

0.918
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **

1.8.1.9
 * *EC#: **

http://www.brenda-enzymes.org/enzyme.php?ecno=1.8.1.9 Continuous Spectrophotometric Rate Determination
 * Link to BRENDA EC# page: **
 * -- ** Show screenshot of BRENDA enzyme mechanism schematic
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/thioredoxinred.pdf
 * -- link to Sigma (or other company ) page for assay (see Sigma links below) **

n/a
 * -- -or link (or citation) to paper that contains assay information **

>>
 * -- links to assay reagents (substrates) pages. **
 * 0.1% (w/v) Thioredoxin Solution (Thioredoxin)
 * http://www.sigmaaldrich.com/catalog/product/sigma/t0910?lang=en®ion=US
 * 100 mM 5'5-Dithio-bis(2-Nitrobenzoic Acid Solution) (DTNB)
 * http://www.sigmaaldrich.com/catalog/product/sigma/d8130?lang=en®ion=US


 * --- List cost and quantity of substrate reagents, supplier, and catalog # **
 * <span style="font-family: 'Courier New',Courier,monospace;">0.1% (w/v) Thioredoxin Solution (Thioredoxin)
 * <span style="font-family: 'Courier New',Courier,monospace;">$190.00/mg, Sigma-Aldrich, T0910
 * <span style="font-family: 'Courier New',Courier,monospace;">100 mM 5'5-Dithio-bis(2-Nitrobenzoic Acid Solution) (DTNB)
 * <span style="font-family: 'Courier New',Courier,monospace;">$31.70/g, Sigma-Aldrich, D8130


 * Structure (PDB or Homology model) **

-- PDB # or closest PDB entry if using homology model: <span style="font-family: 'Courier New',Courier,monospace;">2A87

-- For Homology Model option: n/a Show pairwise alignment of your BLASTP search in NCBI against the PDB n/a Query Coverage: n/a Max % Identities: n/a % Positives n/a Chain used for homology: n/a

<span style="background-color: #ffffff; font-family: 'Times New Roman',stixgeneral,serif; font-size: 15.9990997314453px;">Protoporphyrin IX, (-)-epicatechin-gallate (ECG), (-)-epigallocatechin-3-gallate (EGCG); however, these were tested on rats and calves.
 * Current Inhibitors: **

Yes.
 * Expression Information (has it been expressed in bacterial cells): **

Various affinity chromatography techniques. His-tag-proteins by Ni–NTA column chromatography is a known successful technique for Trxb2.
 * Purification Method : **


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

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

ALRFGADLRMEDVESVSLHGPLKSVVTADGQTHRARAVILAMGAAARYLQVPGEQELLGRGVSSCATCDGFFFRDQDIAV

IGGGDSAMEEATFLTRFARSVTLVHRRDEFRASKIMLDRARNNDKIRFLTNHTVVAVDGDTTVTGLRVRDTNTGAETTLP

VTGVFVAIGHEPRSGLVREAIDVDPDGYVLVQGRTTSTSLPGVFAAGDLVDRTYRQAVTAAGSGCAAAIDAERWLAEHAA

TGEADSTDALIGAQR

335 residues
 * *length of your protein in Amino Acids **

code 35.643 kDa code code Ext. coefficient   14565 Abs 0.1% (=1 g/l)  0.409, assuming all pairs of Cys residues form cystines
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website **
 * Molar Extinction coefficient of your protein at 280 nm wavelength: **

Ext. coefficient   14440 Abs 0.1% (=1 g/l)  0.405, assuming all Cys residues are reduced code

code <span style="background-color: #ffffff; font-family: monospace,serif;"><span class="ff_line">ATGACCGCCCCGCCTGTCCATGACCGCGCACACCACCCCGTTCGCGACGTGATCGTTATCGGCTCCGGTC CCGCGGGGTACACTGCGGCGCTCTACGCCGCCCGTGCCCAGCTGGCGCCGCTGGTCTTCGAGGGCACGTC TTTCGGCGGCGCGCTGATGACCACCACCGACGTGGAGAACTACCCGGGATTTCGCAACGGCATCACCGGT CCAGAGTTGATGGATGAGATGCGGGAACAGGCGCTGCGATTCGGCGCGGACCTGCGTATGGAAGACGTCG AGTCGGTATCACTTCACGGGCCGCTGAAATCGGTCGTCACCGCCGACGGACAGACCCACCGGGCCCGAGC CGTGATCCTGGCAATGGGCGCAGCGGCACGCTATCTGCAGGTGCCCGGCGAACAGGAATTGCTCGGGCGC GGGGTGAGCTCGTGCGCCACCTGCGACGGATTCTTCTTCCGCGATCAGGACATCGCCGTCATCGGCGGCG GTGACTCGGCAATGGAGGAAGCTACCTTCCTGACCCGATTCGCTCGCAGTGTGACGCTGGTGCATCGCCG CGACGAGTTCCGGGCTTCCAAAATCATGCTCGATCGCGCCCGCAACAACGACAAGATACGGTTCCTCACC AACCACACCGTGGTCGCGGTGGACGGGGACACCACAGTGACCGGCTTGCGGGTACGCGACACCAACACCG GTGCCGAAACCACCCTGCCGGTAACCGGTGTTTTCGTCGCGATCGGCCACGAGCCGCGGTCGGGCTTGGT GCGCGAGGCCATCGACGTCGACCCGGACGGCTACGTGTTGGTGCAGGGGCGTACCACCAGCACCTCACTG CCGGGCGTGTTCGCTGCCGGCGACCTGGTGGATCGCACCTATCGCCAGGCGGTTACCGCAGCGGGCAGTG GCTGCGCCGCGGCTATCGACGCCGAGCGCTGGCTCGCCGAGCACGCAGCAACCGGAGAAGCTGACAGTAC CGACGCATTGATAGGAGCACAACGATGA code
 * 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): **

66.1%
 * *GC% Content for gene: **

<span style="background-color: #ffffff; color: #333333; font-family: 'Helvetica Neue',Helvetica,Arial,sans-serif; font-size: 12px;">ATGACGGCGC CACCGGTCCA CGACCGTGCG CACCACCCCG TCCGGGATGT GATCGTGATC GGCTCGGGCC CCGCGGGATA CACCGCGGCC CTGTACGCCG CCCGCGCCCA GCTCGCCCCG CTGGTCTTCG AGGGCACCTC CTTCGGGGGT GCCTTGATGA CCACCACGGA TGTGGAAAAC TACCCGGGCT TTCGCAACGG AATTACCGGC CCGGAGTTGA TGGACGAGAT GCGCGAGCAA GCGCTGCGCT TTGGCGCCGA CCTGCGCATG GAGGATGTGG AATCGGTCTC GCTCCACGGG CCGCTGAAGT CGGTGGTGAC CGCGGACGGC CAGACGCACC GCGCCCGCGC CGTCATCCTG GCCATGGGCG CGGCCGCCCG GTACCTGCAG GTCCCGGGCG AGCAAGAGTT GCTCGGACGC GGCGTGTCGA GCTGCGCGAC CTGTGACGGC TTCTTCTTCC GGGACCAGGA TATCGCGGTG ATCGGCGGAG GTGACTCCGC CATGGAGGAA GCCACCTTCC TGACCCGGTT CGCGCGCTCG GTGACCCTGG TCCACCGGCG GGACGAGTTC CGGGCGTCGA AGATCATGCT CGATCGGGCC CGCAACAATG ACAAGATCCG GTTCCTGACC AATCACACGG TCGTGGCCGT CGACGGCGAT ACGACCGTCA CGGGCTTGCG CGTCCGTGAC ACCAATACCG GCGCCGAGAC CACCTTGCCG GTCACCGGTG TGTTCGTCGC GATCGGCCAC GAGCCGCGCT CGGGGCTGGT GCGCGAGGCG ATCGACGTGG ATCCGGACGG TTATGTCTTG GTGCAAGGTC GGACCACCTC CACGTCGCTG CCCGGCGTGT TCGCGGCCGG CGACCTCGTG GACCGTACCT ACCGCCAAGC GGTGACCGCC GCGGGGTCGG GCTGCGCCGC CGCGATCGAC GCCGAGCGCT GGCTCGCGGA ACACGCGGCG ACCGGAGAAG CCGACTCCAC CGACGCCTTG ATCGGAGCCC AGCGGTAG
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **

69.2%
 * *GC% Content for gene (codon optimized): **

Do Not Need this info for Spring (but still copy these lines to your Target page for now)


 * 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)

-- 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 'tail' primers (this is just **** 2 sequences): **