TargetSp15+-+Acetate+Kinase+(Mycobacterium+Paratuberculosis)

Acetate Kinase (ackA MAP_3886)
 * Target (protein/gene name):**

 [|262316] [ [|NCBI] ]
 * NCBI Gene # or RefSeq#:**

[|NP_962820.1.]
 * Protein ID (NP or XP #) or Wolbachia#:**

 [|Mycobacterium avium subsp. paratuberculosis]
 * Organism (including strain):**

Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
 * Etiologic Risk Group (see link below):**

//Mycobacterium avium //, subspecies //paratuberculosis// (MAP) is a bacterial chronic disease of the intestines. In animals, such as diary cows, the disease presents itself as the Johne’s disease. However, MAP has been traced to be the single etiologic agent of the causation of Crohn’s disease and ulcerative colitis in humans. Classified as an IBD (inflammatory bowel diseases), Crohn’s can cause rectal bleeding, persistent diaherra, fever, and loss of appetite. []
 * Disease Information (sort of like the Intro to your Mini Research Write up):**

[]
 * Link to TDR Targets page (if present):**

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

AckA is essential for catalytic and metabolic activities of e-coli, and renders those two time-dependent without ackA. http://www.ncbi.nlm.nih.gov/pubmed/6252971
 * Essentiality of this protein:**

2 amino acid chains and 4 ligands in ASU
 * Is it a monomer or multimer as biological unit? (make prediction at @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html ):**

“2-mer”
 * Complex of proteins?:**

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

2.7.2.1
 * EC#:**


 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Link to BRENDA EC# page: **<span style="font-family: Arial,sans-serif; font-size: 10pt;">[]

<span style="font-family: Arial,sans-serif; font-size: 10pt;">
 * -- Show screenshot of BRENDA enzyme mechanism schematic**

**<span style="font-family: Arial,sans-serif; font-size: 10pt;">Enzyme Assay ** **<span style="font-family: Arial,sans-serif; font-size: 10pt;">information (spectrophotometric, coupled assay ?, reagents): ** <span style="font-family: Helvetica,sans-serif; font-size: 10.5pt;">Spectrophotometric

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **-- link to Sigma (or other** <span class="apple-converted-space" style="font-family: Arial,sans-serif; font-size: 10pt;"> <span class="r7mb967" style="font-family: Arial,sans-serif; font-size: 10pt;">**company** <span style="font-family: Arial,sans-serif; font-size: 10pt;">**) page for assay (see Sigma links below)** <span style="font-family: Arial,sans-serif; font-size: 10pt;">[]

<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">Acetate activity will be determined by presence of pyruvate with a continuous spectrophotometric rate determination at 340 nm absorbance wavelength. Reagents include 100 mM Triethanolamine Buffer, 1 M Sodium Acetate Solution (NaOAc), 91 mM Adenosine 5'-Triphosphate Solution (ATP), 56 mM Phospho(enol)pyruvate Solution (PEP), 200 mM Magnesium Chloride Solution (MgCl2), 6.4 mM ß-Nicotinamide Adenine Dinucleotide, Reduced Form Solution (ß-NADH), PK/LDH Enzymes Suspension, Myokinase Enzyme Solution (MK), and Acetate Kinase Enzyme Solution. “One unit will phosphorylate 1.0 µmole of acetate to acetyl phosphate per minute at pH 7.6 at 25°C.” =**<span style="color: windowtext; font-family: Calibri,sans-serif; font-size: 11pt;">Structure (PDB or Homology model) **= =<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">3R9P =
 * <span style="color: windowtext; font-family: Calibri,sans-serif; font-size: 11pt;">-- PDB # or closest PDB entry if using homology model: **

=<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Current Inhibitors:** = =<span style="font-family: Arial,sans-serif; font-size: 10pt;">trifluoroethanol (TFE), trifluoroethyl butyrate, N-ethylmaleimide = <span style="font-family: Arial,sans-serif; font-size: 10pt;">[] <span style="font-family: Arial,sans-serif; font-size: 10pt;">[]

<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">Expressed in //E. coli// cells
 * Expression Information (has it been expressed in bacterial cells):**

<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">Ammonium sulfate fractionation <span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">Gel filtration <span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">Diethylaminoethyl-cellulose chromatography <span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">Affinity chromatography on 8-(6-aminohexylamino)-adenosine 5'-triphosphate conjugated to Sepharose 4B []
 * Purification Method:**

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Image of protein (PyMol with features delineated and shown separately):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> <span style="font-family: Arial,sans-serif; font-size: 10pt;"> MDGSDGARRVLVINSGSSSLKFQLVDPEFGVAASTGIVERIGEESSPVPDHDAALRRAFD MLAGDGVDLNTAGLVAVGHRVVHGGNTFYRPTVLDDAVIARLHELSELAPLHNPPALQGI EVARRLLPDIAHVAVFDTGFFHDLPPAAATYAIDRELADRWQIRRYGFHGTSHRYVSEQA AAFLDRPLRGLKQIVLHLGNGCSASAIAGTRPLDTSMGLTPLEGLVMGTRSGDIDPSIVS YLCHTAGMGVDDVESMLNHRSGVVGLSGVRDFRRLRELIESGDGAAQLAYSVFTHRLRKY IGAYLAVLGHTDVISFTAGIGENDAAVRRDAVSGMEELGIVLDERRNLAGGKGARQISAD DSPITVLVVPTNEELAIARDCVRVLGG
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**

code code code code code
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">*length of your protein in Amino Acids: **<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">386
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website **<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">41194.6
 * <span style="font-family: Calibri,sans-serif; font-size: 11pt;">Molar Extinction coefficient of your protein at 280 nm wavelength: **<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">17545

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


 * <span style="font-family: Arial,sans-serif; font-size: 10pt; line-height: 1.5;">*CDS Gene Sequence (paste as text only): **

atggatggcagcgatggcgcgcgccgcgtgctggtgattaacagcggcagcagcagcctg aaatttcagctggtggatccggaatttggcgtggcggcgagcaccggcattgtggaacgc attggcgaagaaagcagcccggtgccggatcatgatgcggcgctgcgccgcgcgtttgat atgctggcgggcgatggcgtggatctgaacaccgcgggcctggtggcggtgggccatcgc gtggtgcatggcggcaacaccttttatcgcccgaccgtgctggatgatgcggtgattgcg cgcctgcatgaactgagcgaactggcgccgctgcataacccgccggcgctgcagggcatt gaagtggcgcgccgcctgctgccggatattgcgcatgtggcggtgtttgataccggcttt tttcatgatctgccgccggcggcggcgacctatgcgattgatcgcgaactggcggatcgc tggcagattcgccgctatggctttcatggcaccagccatcgctatgtgagcgaacaggcg gcggcgtttctggatcgcccgctgcgcggcctgaaacagattgtgctgcatctgggcaac ggctgcagcgcgagcgcgattgcgggcacccgcccgctggataccagcatgggcctgacc ccgctggaaggcctggtgatgggcacccgcagcggcgatattgatccgagcattgtgagc tatctgtgccataccgcgggcatgggcgtggatgatgtggaaagcatgctgaaccatcgc agcggcgtggtgggcctgagcggcgtgcgcgattttcgccgcctgcgcgaactgattgaa agcggcgatggcgcggcgcagctggcgtatagcgtgtttacccatcgcctgcgcaaatat attggcgcgtatctggcggtgctgggccataccgatgtgattagctttaccgcgggcatt ggcgaaaacgatgcggcggtgcgccgcgatgcggtgagcggcatggaagaactgggcatt gtgctggatgaacgccgcaacctggcgggcggcaaaggcgcgcgccagattagcgcggat gatagcccgattaccgtgctggtggtgccgaccaacgaagaactggcgattgcgcgcgat tgcgtgcgcgtgctgggcggc

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***GC% Content for gene:** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***GC% Content for gene (codon optimized):** <span style="font-family: Arial,sans-serif; font-size: 10pt;">Do Not Need this info for Spring (but still copy these lines to your Target page for now) <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):** <span style="font-family: Arial,sans-serif; font-size: 10pt;"> **(** **<span style="color: #008000; font-family: Arial,sans-serif; font-size: 10pt;">link to DNA Works output text file - **<span style="font-family: Arial,sans-serif; font-size: 10pt;">that should be saved in your Google Docs folder after you did the primer design protocol) <span style="font-family: Arial,sans-serif; font-size: 10pt;">-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Primer design results for 'tail' primers (this is just 2 sequences):**