*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.

Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=

-- Show screenshot of BRENDA enzyme mechanism schematic
Fig 1. Enzyme mechanism schematic of AEK1

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
-- link to Sigma (or other company) page for assay (see Sigma links below)

-- -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
-- links to assay reagents (substrates) pages. http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/proteinkinasec.pdf

--- List cost and quantity of substrate reagents, supplier, and catalog #

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

Structure (PDB or Homology model)

-- PDB # or closest PDB entry if using homology model: 5EW9

-- For Homology Model option:

---- Show pairwise alignment of your BLASTP search in NCBI against the PDB

Fig. 2 AEK1 homology models

---- Query Coverage: 90

---- Max % Identities: 4

---- % Positives: 2

---- Chain used for homology: Chain A

Current Inhibitors: No known inhibitors

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.

Purification Method: No purification method has been released by this protein. However, it is important to note that methionine, leucine or phenylalanine can be used in purification.

Image of protein (PyMol with features delineated and shown separately):
Fig 3. AEK1 PyMol image


*length of your protein in Amino Acids: 246

Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 31327.0
Molar Extinction coefficient of your protein at 280 nm wavelength: .98

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

TMPred Chart.PNG
Fig. 4 Tmpred graph of AEK1

*CDS Gene Sequence (paste as text only): ggcccgggcagcaaaaaacgccagtgggcgctggaagattttgaaattggccgcccgctg ggcaaaggcaaatttggcaacgtgtatctggcgcgcgaaaaacagagcaaatttattctg

*GC% Content for gene: 80%

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

*GC% Content for gene (codon optimized): 90%

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):