AEK1+(Trypanosoma+brucei)


 * *Target (protein/gene name): **AEK1

 ***NCBI Gene # or RefSeq#:** 2117786

 ***Protein ID (NP or XP #) or Wolbachia#:** I78840

 ***Organism (including strain):** //Trypanosoma brucei//

**Etiologic Risk Group (see link below):** 3

 ***/Disease Information (sort of like the Intro to your Mini Research Write up):** African trypanosomiasis (also known as African Sleeping Sickness) can be spread to humans by the tsetse fly. Symptoms are pain throughout the body, itching, fever, etc. Later on, patients struggle with changes in behavior, poor hand eye coordination, and confusion. The parasite that causes this disease if found in rural parts of Africa. As of now, the drugs that can inhibit this disease are those that can kill parasites. The protein, AEK1, has just recently been targeted. Animal trials are still being conducted to further understand this protein.

 **Link to TDR Targets page (if present):** None found. This is a protein kinase found in //Trypanosoma brucei.//

 **Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)** http://www.ncbi.nlm.nih.gov/protein/2117786

 **Essentiality of this protein:** Essential to virulence. This is because this protein is a kinase. Also, the protein's essentiality in bloodstream forms has been proven in "A novel protein kinase is essential in bloodstream //Trypanosoma brucei//."

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

 **Complex of proteins?** No

 **Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):** AEK1 was found to a druggable target in rats. This is proven by the following study:Jensen, B. C.; Booster, N.; Vidadala, R. S. R.; Maly, D. J.; Parsons, M. A Novel Protein Kinase Is Essential in Bloodstream Trypanosoma Brucei. //International Journal for Parasitology//. 2016.  ***EC#:** 2.7.1.37

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Link to BRENDA EC# page:** http://www.brenda-enzymes.org/enzyme.php?ecno=2.7.1.37

<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;">Fig 1. Enzyme mechanism schematic of AEK1

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):** Method is Radiolabelled Stop Reaction. Not a coupled assay. Reagents are Tris HCl Buffer, Calcium Chloride Solution, Magnesium Chloride, Chloroform Solution
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">-- link to Sigma (or other company) page for assay (see Sigma links below) **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **-- -or link (or citation) to paper that contains assay information** http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number-ii.html
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">-- links to assay reagents (substrates) pages. **<span style="font-family: Arial,sans-serif; font-size: 10pt;"> http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/proteinkinasec.pdf

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **--- List cost and quantity of substrate reagents, supplier, and catalog #**

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> About 3 M of $60 Tris HCl Buffer, Supplier: Tocris Biosciences, #1185-53-1/ About 5 M of $32 Calcium Chloride Solution, Supplier: Ward Chem, #10043-52-4/ About 5 of $20 Magnesium Chloride, Supplier: Grainger Industries #7791-18-6/ 2 mg/ml Chlorofoam, Supplier: ThomasNet.com #67-66-3

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Structure (PDB or Homology model)**

<span style="font-family: Arial,sans-serif; font-size: 10pt;">-- PDB # or closest PDB entry if using homology model: 5EW9

<span style="font-family: Arial,sans-serif; font-size: 10pt;">-- For Homology Model option:

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> Show pairwise alignment of your BLASTP search in NCBI against the PDB

<span style="font-family: Arial,sans-serif; font-size: 10pt;">Fig. 2 AEK1 homology models

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> Query Coverage: 90

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> Max % Identities: 4

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> % Positives: 2

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> Chain used for homology: Chain A

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Current Inhibitors:** No known inhibitors

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Expression Information (has it been expressed in bacterial cells):** This has not been expressed in bacterial cells. It is expressed by turning on and off a gene in a given organism.

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Purification Method:** No purification method has been released by this protein. However, it is important to note that <span style="color: #2e2e2e; font-family: Arial,sans-serif; font-size: 10pt;">methionine, leucine or phenylalanine can be used in purification.

<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;">Fig 3. AEK1 PyMol image

<span style="font-family: Arial,sans-serif;"> ***Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):** >5EW9:A|PDBID|CHAIN|SEQUENCE GPGSKKRQWALEDFEIGRPLGKGKFGNVYLAREKQSKFILALKVLFKAQLEKAGVEHQLRREVEIQSHLRHPNILRLYGYFHDATRVYLILEYAPLGTVYRELQKLSKFDEQRTATYITELANALSYCHSKRVIHRDIKPENLLLGSAGELKIADFGWSVHAPSSRRTTLCGTLDYLPPEMIEGRMHDEKVDLWSLGVLCYEFLVGKPPFEANTYQETYKRISRVEFTFPDFVTEGARDL ISRLLKHNPSQRPMLREVLEHPWITANSSKP

<span style="font-family: Arial,sans-serif;"> ***length of your protein in Amino Acids:** 246

<span style="font-family: Arial,sans-serif;"> **Molecular Weight of your protein in kiloDaltons using the** [|**Expasy ProtParam**]**<span style="font-family: Arial,sans-serif;"> website: **<span style="font-family: Arial,sans-serif;">31327.0
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Molar Extinction coefficient of your protein at 280 nm wavelength: **<span style="font-family: Arial,sans-serif; font-size: 10pt;">.98

<span style="font-family: Arial,sans-serif; font-size: 10pt;">**TMpred graph Image** ( <span style="color: #0000ff; font-family: Arial,sans-serif; font-size: 10pt;">@http://www.ch.embnet.org/software/TMPRED_form.html <span style="font-family: Arial,sans-serif; font-size: 10pt;">). Input your amino acid sequence to it.

<span style="font-family: Arial,sans-serif; font-size: 10pt; line-height: 1.5;">Fig. 4 Tmpred graph of AEK1

<span style="font-family: Arial,sans-serif; font-size: 10pt;">gcgctgaaagtgctgtttaaagcgcagctggaaaaagcgggcgtggaacatcagctgcgc <span style="font-family: Arial,sans-serif; font-size: 10pt;">cgcgaagtggaaattcagagccatctgcgccatccgaacattctgcgcctgtatggctat <span style="font-family: Arial,sans-serif; font-size: 10pt;">tttcatgatgcgacccgcgtgtatctgattctggaatatgcgccgctgggcaccgtgtat <span style="font-family: Arial,sans-serif; font-size: 10pt;">cgcgaactgcagaaactgagcaaatttgatgaacagcgcaccgcgacctatattaccgaa <span style="font-family: Arial,sans-serif; font-size: 10pt;">ctggcgaacgcgctgagctattgccatagcaaacgcgtgattcatcgcgatattaaaccg <span style="font-family: Arial,sans-serif; font-size: 10pt;">gaaaacctgctgctgggcagcgcgggcgaactgaaaattgcggattttggctggagcgtg <span style="font-family: Arial,sans-serif; font-size: 10pt;">catgcgccgagcagccgccgcaccaccctgtgcggcaccctggattatctgccgccggaa <span style="font-family: Arial,sans-serif; font-size: 10pt;">atgattgaaggccgcatgcatgatgaaaaagtggatctgtggagcctgggcgtgctgtgc <span style="font-family: Arial,sans-serif; font-size: 10pt;">tatgaatttctggtgggcaaaccgccgtttgaagcgaacacctatcaggaaacctataaa <span style="font-family: Arial,sans-serif; font-size: 10pt;">cgcattagccgcgtggaatttacctttccggattttgtgaccgaaggcgcgcgcgatctg <span style="font-family: Arial,sans-serif; font-size: 10pt;">attagccgcctgctgaaacataacccgagccagcgcccgatgctgcgcgaagtgctggaa <span style="font-family: Arial,sans-serif; font-size: 10pt;">catccgtggattaccgcgaacagcagcaaaccg
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">*CDS Gene Sequence (paste as text only): **<span style="font-family: Arial,sans-serif; font-size: 10pt;">ggcccgggcagcaaaaaacgccagtgggcgctggaagattttgaaattggccgcccgctg ggcaaaggcaaatttggcaacgtgtatctggcgcgcgaaaaacagagcaaatttattctg

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***GC% Content for gene:** 80%

<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;">ggcccgggcagcaaaaaacgccagtgggcgctggaagattttgaaattggccgcccgctgggcaaaggcaaatttggcaacgtgtatctggcgcgcgaaaaacagagcaaatttattctggcgctgaaagtgctgtttaaagcgcagctggaaaaagcgggcgtggaacatcagctgcgccgcgaagtggaaattcagagccatctgcgccatccgaacattctgcgcctgtatggctattttcatgatgcgacccgcgtgtatctgattctggaatatgcgccgctgggcaccgtgtatcgcgaactgcagaaactgagcaaatttgatgaacagcgcaccgcgacctatattaccgaactggcgaacgcgctgagcattgccatagcaaacgcgtgattcatcgcgatattaaaccggaaaacctgctgctgggcagcgcgggcgaactgaaaattgcggattttggctggagcgtgcatgcgccgagcagccgccgcaccaccctgtgcggcaccctggattatctgccgccggaaatgattgaaggccgcatgcatgatgaaaaagtggatctgtggagcctgggcgtgctgtgctatgaatttctggtgggcaaaccgccgtttgaagcgaacacctatcaggaaacctataaacgcattagccgcgtggaatttacctttccggattttgtgaccgaaggcgcgcgcgatctgattagccgcctgctgaaacataacccgagccagcgcccgatgctgcgcgaagtgctggaacatccgtggattaccgcgaacagcagcaaaccg

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***GC% Content for gene (codon optimized):** 90%

<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):**

<span style="font-family: Arial,sans-serif; font-size: 10pt;">**