Target-+3D7,+glutathione+reductase


 * Dr. B Notes 090613 **
 * do gel extractin on PCR squared **
 * crystal structures don't have Glutathione nor NADPH (they do have FAD) **
 * What is the role of FAD - is it needed for catalysis, or is it just a 'space filler'/crystallization reagent. **
 * How can we get NADPH in the active site for docking? **
 * How can we define the Glutathione area for docking? **


 * *Target (protein/gene name): **P. falciparum 3D7, glutathione reductase
 * *NCBI Gene # or RefSeq#: **AF027825.1
 * *Protein ID (NP or XP #) or Wolbachia#: **AAB84117.1
 * *Organism (including strain): Plasmodium falciparum **
 * *Etiologic Risk Group (see link below): Risk Group 2 (RG2) - Parasitic Agents **
 * *Background/Disease Information (sort of like the Intro to your Mini Research Write up): **

Plasmodium falciparum is a protozoan parasite, one of the species of Plasmodium that cause malaria in humans. It is transmitted by the female Anopheles mosquito. Also, //Plasmodium falciparum// is the most deadly of the five //Plasmodium// species. Malaria has a massive impact on human health; it is the world’s second biggest killer after tuberculosis. Around 300 million clinical cases occur each year resulting in between 1.5 - 2.7 million deaths annually, the majority in sub-saharan Africa. It is estimated that 3,000 children under the age of five years fall victim to malaria each day. Around 40% of the world’s population is at risk. Malaria caused by this species (also called malignant or falciparum malaria) is the most dangerous form of malaria, with the highest rates of complications and mortality Almost every malarial death is caused by P. falciparum.

TDR Targets Link: http://www.tdrtargets.org/targets/view?gene_id=2524

[] [] [] GRSA-1KT $579.00 per pack (this is the whole kit - not needed)
 * *Essentiality of this protein: Protein is essential **
 * Gene/Ortholog:** mtu2902 (OG4_10249); **Phenotype:** essential; **Source study:** nmpdr
 * Gene/Ortholog:** eco3379 (OG4_10249); **Phenotype:** essential; **Source study:** gerdes
 * Gene/Ortholog:** Tb927.10.10390 (OG4_10249); **Phenotype:** significant loss of fitness in bloodstream forms (6 days); **Source study:** alsford
 * Gene/Ortholog:** Tb927.10.10390 (OG4_10249); **Phenotype:** significant loss of fitness in differentiation of procyclic to bloodstream forms; **Source study**alsford
 * <span style="font-family: Calibri,sans-serif;">**Complex of proteins?: Works alone**
 * <span style="font-family: 'Calibri','sans-serif';">*Druggable Target: Yes **
 * <span style="font-family: 'Calibri','sans-serif';">*EC#: 1.8.1.7 **
 * <span style="font-family: 'Calibri','sans-serif';">*Link to BRENDA EC# page: **
 * <span style="font-family: 'Calibri','sans-serif';">*Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **
 * <span style="font-family: 'Calibri','sans-serif';">-- link to Sigma (or other company) page for assay or assay reagents (substrates) **
 * <span style="font-family: 'Calibri','sans-serif';">-- link (or citation) to paper that contains assay information **
 * <span style="font-family: 'Calibri','sans-serif';">-- List cost and quantity of substrate reagents and supplier **
 * <span style="font-family: 'Calibri','sans-serif';">[] **

http://www.sigmaaldrich.com/catalog/product/sigma/g4626?lang=en®ion=US
 * G4626** **Sigma** - glutathione

-- PDB # or closest PDB entry if using homology model: 3SQP
 * <span style="font-family: 'Calibri','sans-serif';">*Structure Available (PDB or Homology model) **

Possible to express using E.coli [] Glutathione reductase (E.C.1.8.1.7) was purified from //Phaeodactylum tricornutum// cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2<span style="font-family: 'Calibri','sans-serif';">’ ,5<span style="font-family: 'Calibri','sans-serif';">’ -ADP-Sepharose). []
 * *Current Inhibitors:25 **
 * *Expression Information (has it been expressed in bacterial cells): **
 * *Purification Method: **

<span style="font-family: 'Calibri','sans-serif';"> <span style="font-family: 'Calibri','sans-serif'; line-height: 0px; overflow: hidden;"> >gi|2599403|gb|AAB84117.1| glutathione reductase [Plasmodium falciparum] MVYDLIVIGGGSGGMAAARRAARHNAKVALVEKSRLGGTCVNVGCVPKKIMFNAASVHDILENSRHYGFD TKFSFNLPLLVERRDKYIQRLNNIYRQNLSKDKVDLYEGTASFLSENRILIKGTKDNNNKDNGPLNEEIL EGRNILIAVGNKPVFPPVKGIENTISSDEFFNIKESKKIGIVGSGYIAVELINVIKRLGIDSYIFARGNR ILRKFDESVINVLENDMKKNNINIVTFADVVEIKKVSDKNLSIHLSDGRIYEHFDHVIYCVGRSPDTENL
 * *Image of protein (PyMol with features delineated and shown separately): **
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **

VQLTPVAINAGRLLADRLFLKKTRKTNYKLIPTVIFSHPPIGTIGLSEEAAIQIYGKENVKIYESKFTNL FFSVYDIEPELKEKTYLKLVCVGKDELIKGLHIIGLNADEIVQGFAVALKMNATKKDFDETIPIHPTAAE EFLTLQPWMK

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
 * <span style="font-family: 'Calibri','sans-serif';">*length of your protein in Amino Acids: 500 **
 * <span style="font-family: 'Calibri','sans-serif';">Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: 56419.9 **
 * <span style="font-family: 'Calibri','sans-serif';">Molar Extinction coefficient of your protein at 280 nm wavelength: **
 * Ext. coefficient 34185
 * Abs 0.1% (=1 g/l) 0.606, assuming all pairs of Cys residues form cystines
 * Ext. coefficient 33810
 * Abs 0.1% (=1 g/l) 0.599, assuming all Cys residues are reduced
 * <span style="font-family: 'Calibri','sans-serif';">TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.


 * <span style="font-family: 'Calibri','sans-serif';">*CDS Gene Sequence (paste as text only): **

>gi|2599402|gb|AF027825.1| Plasmodium falciparum glutathione reductase (GR3) mRNA, complete cds AAAGTTTTTTTTTTTTTTTTTCTTATAAAACTTATTAATTATGTACAAACATAGATACTTTCATTTTTTT TCTTTTTCTTTTTTTTTCTCGTGTCAACCAAAATAATAAGAAGTTTTACTTTTCTTAACAATAATACAAA TTTGAGTAATCCAGTATACTTTAAAAAAAACGCAAATATGGTTTACGATTTAATTGTAATTGGTGGTGGA AGTGGAGGAATGGCTGCAGCTAGGAGGGCAGCAAGGCATAACGCAAAAGTTGCTCTTGTCGAAAAATCCC GTTTAGGTGGAACGTGTGTCAACGTTGGATGTGTTCCTAAAAAAATTATGTTCAATGCTGCCTCAGTTCA TGATATTTTGGAAAATTCCAGGCATTACGGATTTGACACCAAATTTTCGTTCAACTTACCTCTGTTGGTA GAGAGAAGGGATAAGTACATTCAAAGGTTGAATAATATTTATAGACAGAATTTAAGTAAGGATAAGGTAG ATTTGTACGAAGGAACAGCTAGCTTTTTAAGCGAAAATAGGATATTAATAAAAGGCACAAAAGATAATAA TAATAAGGATAATGGGCCTTTGAATGAAGAAATCCTTGAAGGAAGAAATATTCTTATAGCTGTTGGAAAT AAACCAGTATTTCCACCGGTTAAAGGTATAGAAAATACAATATCAAGTGATGAATTTTTTAATATTAAAG AATCTAAAAAAATTGGTATTGTTGGAAGTGGATATATAGCTGTTGAATTAATAAATGTTATAAAAAGATT AGGTATTGATTCTTATATATTTGCAAGAGGAAATAGAATATTAAGGAAATTTGATGAGTCTGTTATTAAT GTATTAGAAAATGATATGAAAAAGAATAATATAAATATTGTAACATTTGCAGATGTTGTTGAAATAAAAA AAGTTAGTGATAAAAATTTGTCCATACATTTATCTGATGGAAGAATATATGAACATTTTGATCATGTCAT TTATTGTGTTGGTAGATCACCAGATACCGAAAATTTAAATTTAGGAAAATTAAATGTGGAAACAAATAAT AATTATATAGTAGTAGATGAAAATCAACGAACTAGTGTAAACAATATTTATGCTGTTGGTGATTGTTGTA TGGTAAAAAAATCAAAAGAAATTGAAGATTTAAATTTATTGAAATTATATAATGAAGAAACATATCTAAA TAAAAAAGAAAATGTTACAGAAGATATTTTTTATAATGTACAATTAACACCGGTAGCTATTAATGCAGGA AGATTATTAGCTGATAGATTATTTTTAAAAAAAACACGAAAAACAAATTATAAACTTATACCAACTGTTA TATTTTCTCATCCACCTATAGGTACTATTGGTCTTTCTGAAGAAGCAGCAATTCAAATATATGGAAAGGA AAATGTTAAAATATATGAATCCAAATTTACTAATTTATTCTTTTCAGTTTATGATATAGAACCAGAACTA AAAGAAAAAACATATCTTAAATTAGTATGTGTTGGAAAAGATGAATTAATTAAAGGATTACATATAATAG GATTAAATGCAGATGAAATTGTTCAAGGTTTTGCAGTGGCCTTAAAAATGAATGCTACCAAAAAGGATTT CGATGAAACCATACCTATACATCCTACAGCAGCAGAAGAATTTCTAACCTTACAGCCATGGATGAAATGA GTTCTCTCTTGAATAACTTCTTCACATAGGATTGTGTATATGTAATATATTAATATATATTAATAAATAA TTAGTATATCATGCGTTTAAGCTGGCATATCTTAAAAGTTGTATAAATATCATATACAAGCGCGCGCGCG CCCATACATACATACATAAATAAATATATATATATATTAATTTATTTTATTAGAACACCTTATGTTTCCT AGCTATAATTCATATTACCCTTTAATCGTATTTATTTTTTTATTTTTTTATTTTTTTTATTTCTTTTTAA TATTCTTCAAACCATTATAGTAAACCTACATATCCTTTCACATTTATACTCAAAGTGGTACCTTGTGAAT TTTTATTTTTATTTTTTAAATCAACACATATACTACAATAAATTGTAAAAACTTTATAATATTAAATATT TTATTTATTTTTAAAAAAAAAAAAAAAA


 * <span style="font-family: 'Calibri','sans-serif';">*GC% Content for gene: 25.8% **

1 ATGGTCTACGATCTGATTGTAATTGGTGGTGGCTCTGGTGGTATGGCGGCTGCACGCCGT 61 GCCGCACGTCACAACGCGAAAGTTGCACTGGTTGAAAAATCCCGTCTGGGTGGTACCTGT 121 GTGAACGTGGGCTGCGTGCCGAAAAAGATCATGTTCAATGCGGCGTCTGTTCATGACATC 181 CTCGAGAACTCTCGCCACTATGGTTTTGATACGAAATTCTCCTTCAATCTCCCACTGCTG 241 GTCGAACGTCGTGACAAATACATCCAGCGTCTCAACAACATTTATCGCCAGAACCTCTCT 301 AAAGACAAGGTTGACCTGTACGAAGGCACCGCGTCTTTCCTCTCTGAAAACCGTATTCTG 361 ATTAAAGGTACGAAAGATAATAACAACAAGGACAATGGTCCGCTGAACGAGGAAATCCTG 421 GAAGGTCGTAACATCCTGATCGCGGTCGGTAACAAACCTGTTTTCCCACCGGTAAAAGGT 481 ATCGAAAACACCATCAGCAGCGACGAGTTCTTCAACATCAAGGAATCCAAGAAAATCGGT 541 ATCGTTGGTAGCGGTTATATCGCAGTCGAACTGATCAACGTGATCAAACGCCTCGGTATT 601 GACTCTTACATCTTCGCGCGTGGTAACCGCATTCTGCGTAAGTTTGACGAGTCTGTCATC 661 AATGTTCTGGAAAATGATATGAAGAAGAATAACATTAACATCGTTACCTTCGCGGACGTT 721 GTAGAAATCAAGAAAGTCTCTGACAAAAATCTGTCTATTCACCTGAGCGACGGTCGCATC 781 TATGAACACTTTGACCACGTCATTTACTGCGTCGGTCGTTCCCCGGATACTGAGAATCTC 841 AACCTCGAAAAACTGAACGTTGAAACGAACAATAACTATATCGTGGTCGACGAGAACCAG 901 CGTACGTCCGTAAACAATATCTATGCGGTAGGCGACTGCTGCATGGTTAAAAAGTCTAAG 961 GAGATCGAAGACCTGAATCTGCTCAAGCTGTACAACGAAGAGACGTATCTGAACAAAAAA 1021 GAGAACGTCACCGAGGACATCTTTTACAATGTCCAGCTGACCCCAGTTGCCATCAACGCG 1081 GGTCGTCTGCTGGCGGATCGCCTCTTCCTGAAAAAAACCCGTAAGACCAACTACAAGCTG 1141 ATTCCAACCGTGATTTTTAGCCACCCGCCAATCGGCACTATTGGTCTGTCTGAGGAAGCT 1201 GCGATCCAGATCTACGGTAAAGAAAACGTTAAAATCTACGAATCTAAATTCACCAACCTC 1261 TTCTTTTCTGTTTATGACATTGAGCCGGAGCTGAAGGAAAAGACCTACCTGAAGCTGGTT 1321 TGTGTTGGCAAAGACGAACTCATTAAGGGTCTGCACATCATCGGTCTGAACGCCGACGAA 1381 ATCGTTCAAGGTTTCGCCGTGGCGCTCAAGATGAACGCGACGAAGAAGGACTTCGATGAA 1441 ACCATCCCGATCCACCCGACGGCGGCAGAAGAGTTCCTCACTCTGCAGCCATGGATGAAA 1501 TAA
 * <span style="font-family: 'Calibri','sans-serif';">*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **


 * <span style="font-family: 'Calibri','sans-serif';">*GC% Content for gene (codon optimized): 47% **

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


 * <span style="font-family: 'Calibri','sans-serif';">Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): **


 * <span style="font-family: 'Calibri','sans-serif';">( <span style="color: #008000; font-family: 'Calibri','sans-serif';">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.

1 ATGGTCTACGATCTGATTGTAATTGGTGGTGGCTCTGGTGGTATGGCGGCTGCACGC 57 2 GGGATTTTTCAACCAGTGCAACTTTCGCGTTGTGACGTGCGGCACGGCGTGCAGCCGCCA 60 3 TTGCACTGGTTGAAAAATCCCGTCTGGGTGGTACCTGTGTGAACGTGGGCTGCGTGCCGA 60 4 TTCTCGAGGATGTCATGAACAGACGCCGCATTGAACATGATCTTTTTCGGCACGCAGCCC 60 5 TGTTCATGACATCCTCGAGAACTCTCGCCACTATGGTTTTGATACGAAATTCTCCTTCAA 60 6 TGTATTTGTCACGACGTTCGACCAGCAGTGGGAGATTGAAGGAGAATTTCGTATCAAAAC 60 7 TCGAACGTCGTGACAAATACATCCAGCGTCTCAACAACATTTATCGCCAGAACCTCTCTA 60 8 AAAGACGCGGTGCCTTCGTACAGGTCAACCTTGTCTTTAGAGAGGTTCTGGCGATAAATG 60 9 GAAGGCACCGCGTCTTTCCTCTCTGAAAACCGTATTCTGATTAAAGGTACGAAAGATAAT 60 10 CCTCGTTCAGCGGACCATTGTCCTTGTTGTTATTATCTTTCGTACCTTTAATCAGAATAC 60 11 TGGTCCGCTGAACGAGGAAATCCTGGAAGGTCGTAACATCCTGATCGCGGTCGGTAACAA 60 12 CTGATGGTGTTTTCGATACCTTTTACCGGTGGGAAAACAGGTTTGTTACCGACCGCGATC 60 13 TAAAAGGTATCGAAAACACCATCAGCAGCGACGAGTTCTTCAACATCAAGGAATCCAAGA 60 14 TCGACTGCGATATAACCGCTACCAACGATACCGATTTTCTTGGATTCCTTGATGTTGAAG 60 15 GCGGTTATATCGCAGTCGAACTGATCAACGTGATCAAACGCCTCGGTATTGACTCTTACA 60 16 CGTCAAACTTACGCAGAATGCGGTTACCACGCGCGAAGATGTAAGAGTCAATACCGAGGC 60 17 GCATTCTGCGTAAGTTTGACGAGTCTGTCATCAATGTTCTGGAAAATGATATGAAGAAGA 60 18 ACAACGTCCGCGAAGGTAACGATGTTAATGTTATTCTTCTTCATATCATTTTCCAGAACA 60 19 ACCTTCGCGGACGTTGTAGAAATCAAGAAAGTCTCTGACAAAAATCTGTCTATTCACCTG 60 20 ACGTGGTCAAAGTGTTCATAGATGCGACCGTCGCTCAGGTGAATAGACAGATTTTTGTCA 60 21 TCTATGAACACTTTGACCACGTCATTTACTGCGTCGGTCGTTCCCCGGATACTGAGAATC 60 22 AGTTATTGTTCGTTTCAACGTTCAGTTTTTCGAGGTTGAGATTCTCAGTATCCGGGGAAC 60 23 TGAACGTTGAAACGAACAATAACTATATCGTGGTCGACGAGAACCAGCGTACGTCCGTAA 60 24 ACTTTTTAACCATGCAGCAGTCGCCTACCGCATAGATATTGTTTACGGACGTACGCTGGT 60 25 ACTGCTGCATGGTTAAAAAGTCTAAGGAGATCGAAGACCTGAATCTGCTCAAGCTGTACA 60 26 CGGTGACGTTCTCTTTTTTGTTCAGATACGTCTCTTCGTTGTACAGCTTGAGCAGATTCA 60 27 ACAAAAAAGAGAACGTCACCGAGGACATCTTTTACAATGTCCAGCTGACCCCAGTTGCCA 60 28 TTTTTCAGGAAGAGGCGATCCGCCAGCAGACGACCCGCGTTGATGGCAACTGGGGTCAGC 60 29 GGATCGCCTCTTCCTGAAAAAAACCCGTAAGACCAACTACAAGCTGATTCCAACCGTGAT 60 30 CTCAGACAGACCAATAGTGCCGATTGGCGGGTGGCTAAAAATCACGGTTGGAATCAGCTT 60 31 GGCACTATTGGTCTGTCTGAGGAAGCTGCGATCCAGATCTACGGTAAAGAAAACGTTAAA 60 32 AAAAGAAGAGGTTGGTGAATTTAGATTCGTAGATTTTAACGTTTTCTTTACCGTAGATCT 60 33 TCTAAATTCACCAACCTCTTCTTTTCTGTTTATGACATTGAGCCGGAGCTGAAGGAAAAG 60 34 ATGAGTTCGTCTTTGCCAACACAAACCAGCTTCAGGTAGGTCTTTTCCTTCAGCTCCGGC 60 35 TGTTGGCAAAGACGAACTCATTAAGGGTCTGCACATCATCGGTCTGAACGCCGACGAAAT 60 36 TCGTCGCGTTCATCTTGAGCGCCACGGCGAAACCTTGAACGATTTCGTCGGCGTTCAGAC 60 37 CTCAAGATGAACGCGACGAAGAAGGACTTCGATGAAACCATCCCGATCCACCCGACGGCG 60 38 TTATTTCATCCATGGCTGCAGAGTGAGGAACTCTTCTGCCGCCGTCGGGTGGAT 54

Forward Tail Primer: TACTTCCAATCCATGGTCTACGATCTGATTGTAAT
 * <span style="font-family: 'Calibri','sans-serif';">Primer design results for 'tail' primers (this is just 2 sequences): **

Reverse Tail Primer: __<span style="font-family: 'Calibri','sans-serif'; font-size: 16px;">CTCTGCAGCCATGGATGAAA __<span style="font-family: 'Calibri','sans-serif'; font-size: 16px;">TAA <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">CAGTAAAGGTGGATA

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

@http://www.niaid.nih.gov/Pages/default.aspx @http://eupathdb.org/eupathdb/ @https://patricbrc.vbi.vt.edu/portal/portal/patric/Home @http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes @http://tubic.tju.edu.cn/deg/ @http://csgid.org/csgid/cake/pages/community_request_gateway @http://tdrtargets.org/ @http://gsc.jcvi.org/status.shtml
 * 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 **


 * Is my target good for Virtual Screening programs? **