Target+-+Aspartate+Aminotransferase.+Leishmania+major+(Friedlin+strain)

***Target (protein/gene name): P** utative Aspartate Aminotransferase

 * *NCBI Gene # or RefSeq#: **5652203


 * *Protein ID (NP or XP #) or Wolbachia#: **LmjF.24.0370


 * *Organism (including strain): **// Leishmania major strain Friedlin //


 * Etiologic Risk Group (see link below): **Risk group 2 (RG2) - parasitic agents

Leishmania major is a species of protozoa that cause zoonotic cutaneous leishmaniasis by infecting the macrophages and dendritic cells of the immune system. It is mostly found in Northwestern India, Northern Africa, and Northwestern China. It is transmitted by female sand flies and reproduces through binary fission once engulfed by the host’s macrophages. Symptoms of the disease involve lesions at the bitten areas and lasts around 3-6 months. While there is no cure yet, treatments include Fluconazole which can speed up recovery.
 * *Background/Disease Information (sort of like the Intro to your Mini Research Write up): **


 * Link to TDR Targets page (if present): **http://tdrtargets.org/targets/view?gene_id=29531


 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): **http://www.ncbi.nlm.nih.gov/gene/5652203

Aspartate aminotransferases are involved in the final step of methionine regeneration from methylthioadenosine. Methionine is required for the initiation of protein synthesis, the biosynthesis of cysteine, phospholipids, and polyamines. It is also used for the methylation of rRNA and xenobiotics. Rapidly growing cells (which include those of parasites) synthesize large amounts of polyamines before DNA replication and inhibiting the formation of methionine can inhibit this process. In addition, since methionine is always the first amino acid in protein synthesis, a lack of methionine can inhibit this process as well.
 * Essentiality of this protein: **

LmjF24.0370 has essentiality data

Gene/Ortholog: eco898 (OG4_10189); Phenotype: non-essential; Source study: blattner

Gene/Ortholog: eco898 (OG4_10189); Phenotype: non-essential; Source study: gerdes

Gene/Ortholog: eco898 (OG4_10189); Phenotype: non-essential; Source study: keio

Gene/Ortholog: eco898 (OG4_10189); Phenotype: non-essential; Source study: shigen

Gene/Ortholog: eco3899 (OG4_10189); Phenotype: non-essential; Source study: blattner

Gene/Ortholog: eco3899 (OG4_10189); Phenotype: essential; Source study: gerdes

Gene/Ortholog: eco3899 (OG4_10189); Phenotype: non-essential; Source study: keio

Gene/Ortholog: eco3899 (OG4_10189); Phenotype: non-essential; Source study: shigen

Gene/Ortholog: cel14208 (OG4_10189); Phenotype: Growth Defect; Source study: neb

Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in bloodstream forms (3 days); Source study: alsford

Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: significant gain of fitness in bloodstream forms (6 days); Source study: alsford

Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in procyclic forms; Source study: alsford

Gene/Ortholog: Tb927.10.3660 (OG4_10189); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford

Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford

Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: no significant loss or gain of fitness in bloodstream forms (6 days); Source study: alsford

Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford

Gene/Ortholog: Tb11.02.2740 (OG4_10189); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford


 * Complex of proteins?: ** No

//Mus musculus//: http://www.ncbi.nlm.nih.gov/pubmed/11101353
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **


 * *EC#: **2.6.1.1


 * Link to BRENDA EC# page: **http://www.brenda-enzymes.org/php/result_flat.php4?ecno=2.6.1.1
 * -- ** Show screenshot of BRENDA enzyme mechanism schematic




 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): ** Assay type: Spectrophotometric


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


 * -- links to assay reagents (substrates) pages. **http://www.ncbi.nlm.nih.gov/pubmed/7379304


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

2 pack - Reaction Cocktail containing Reconstitute AST 10 Reagent, Stock No. 58-10, in 10 ml of deionized water ($96.82, ThermoScientific, TR70121)

1 pack - Type I, ammonium sulfate suspension, 200-500 units/mg protein, ($61.40, Sigma Aldrich, G2751-1KU)


 * Structure Available (PDB or Homology model): ** PDB #: 4H51


 * Current Inhibitors: ** Canaline, Carboxymethoxylamine


 * Expression Information (has it been expressed in bacterial cells): ** It has been expressed in //E. Coli// cells before.


 * Purification Method: ** Affinity chromatography - A solution containing 0.02 M potassium phosphate buffer with 250-300 mg of the enzyme is added to the hydroxyapatite column and eluted with the potassium phosphate buffer.


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



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

CIGVYRDEQNKPFVLESVRKAMSHIVERDTQMDYAPIAGLPSFVNSVQRLCFGKPMLDVQ

GDRIASAQTLSGTGALHLGVQLLQRSSGGSGTATLHIPSPSYPNHLNILQHLNVEASYYP

YYNLNTHRLNIEAMLNYLRQLPAGSVVLLHACAHNPTGCDPTPEEWQQIVDVICRSDLIP

FVDMAYQGFATGDIERDAYVLRALNQQEVPTYLVAQSLAKSFGLYGHRTGAVHIRCTTQK

EKANVLSQLQSTIRATYSNPPIFGARIADEILRTPQLRELWKSELNQMSSRLQDVRHRLV

AQLRACGSTRDWEYLEKGVGMMSLTGLTEEQVMALQQKYSVYLTRNGRIAFSGLSSENVA

YVAQSIHDVSR


 * *length of your protein in Amino Acids: ** 431


 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: **47.653 kDa


 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 44810 M-1 cm-1


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

1 atgcttcgtc acgtgtgctc gtccgccgtc gtggcctaca gtgctggcct ggcgccggtc 61 agctgccgcc gtgacgggtc gacgagctac ttctcggccg tgccgcgcgc gcctccagat 121 gccatcatgg gcattgctgc ggactttgcc aaggacatgt gcccaagcaa ggtaaacctt 181 tgcatcggag tctaccgtga tgagcagaac aagccctttg tgctggagtc tgtgcgcaag 241 gccatgtcgc acattgtgga gcgcgacacg cagatggact atgcacccat tgcgggtctg 301 ccgtccttcg tgaatagcgt gcagcggcta tgctttggaa agccaatgct ggacgttcaa 361 ggggaccgca tcgcctcggc acaaactctt agtggcaccg gtgccctgca cctcggcgtg 421 cagctactgc agcgctcctc tggtggctcc ggcaccgcga cgctgcacat cccgagcccc 481 tcctacccga atcacctgaa tatcctgcag catctaaacg ttgaggctag ctattacccg 541 tactacaatc tgaacacgca tcgcttgaac atcgaggcga tgctcaacta tctgcgccag 601 ctgcccgccg gctccgtggt cctgctgcac gcctgtgcgc acaacccgac tgggtgcgac 661 cctactccag aggaatggca gcagattgta gacgtcatct gccgaagcga tctcattccg 721 ttcgttgaca tggcatacca gggttttgcg acgggtgaca tcgagcgtga cgcctacgtc 781 ctacgcgcgt tgaatcagca agaggtcccc acgtacttgg tggcccagtc gttggcgaag 841 agctttggtc tctacgggca tcgcactggc gccgtccaca tccgctgcac cacgcagaag 901 gagaaggcaa atgtcctgtc gcagctgcag tcgacgattc gcgccaccta cagcaaccct 961 cccatcttcg gtgctcgcat cgcggacgaa attttgcgaa ccccgcagct gcgcgagctg 1021 tggaagtcgg agctgaacca gatgtcgagc cgactgcagg atgttcggca ccgcctcgtg 1081 gcccagctgc gtgcttgtgg ctcgacgcgc gactgggagt acctcgagaa aggtgtgggt 1141 atgatgtcgc tgacggggct gacggaggag caggtgatgg cgctgcagca gaagtacagc 1201 gtctacctga cgcgcaacgg tcgcatcgca ttttctggac tgagctccga aaatgtggcc 1261 tacgtcgctc agtccatcca cgatgtgtcc cggtag
 * *CDS Gene Sequence (paste as text only): **


 * *GC% Content for gene: ** 60.57%

ATGCTGCGCC ATGTATGCAG CAGTGCGGTT GTCGCGTATT CCGCAGGTCT GGCCCCGGTG AGTTGCCGTC GCGACGGTAG TACTTCATAC TTCAGCGCGG TTCCGCGGGC ACCGCCGGAT GCCATTATGG GCATTGCCGC CGATTTTGCG AAAGATATGT GCCCGAGTAA GGTCAATCTG TGCATTGGAG TTTACCGCGA CGAACAGAAT AAACCATTTG TGCTGGAAAG TGTCCGGAAA GCGATGTCCC ACATCGTGGA ACGCGATACA CAAATGGATT ATGCTCCTAT TGCGGGGCTG CCCAGTTTTG TTAATAGCGT TCAGCGTCTG TGTTTCGGAA AACCTATGTT AGACGTACAG GGGGATCGCA TCGCCAGTGC TCAGACTCTG AGTGGCACCG GAGCACTGCA CCTGGGCGTT CAGCTTCTCC AACGGAGCTC GGGGGGGTCC GGGACGGCGA CTCTCCATAT CCCATCGCCG AGCTACCCTA ACCACCTTAA TATTCTCCAA CACCTGAACG TAGAGGCGTC GTACTATCCA TACTATAACC TCAACACCCA CCGTCTGAAT ATTGAAGCCA TGCTGAACTA TCTGCGCCAA TTACCGGCCG GCAGTGTTGT GCTGTTGCAT GCGTGTGCGC ACAACCCTAC TGGGTGCGAT CCGACTCCGG AGGAGTGGCA ACAGATCGTC GATGTCATCT GCCGCTCAGA TTTGATCCCG TTCGTCGATA TGGCCTACCA AGGTTTCGCA ACGGGGGATA TCGAACGGGA TGCTTATGTG TTGCGCGCGT TAAACCAGCA GGAAGTACCC ACCTATCTGG TGGCTCAGTC CCTGGCCAAG AGCTTTGGCC TGTACGGGCA TCGGACTGGG GCGGTGCACA TTCGGTGCAC TACACAGAAA GAAAAAGCAA ACGTCCTGTC CCAACTTCAA AGCACCATTC GCGCCACCTA TTCCAATCCC CCGATCTTCG GAGCCCGTAT TGCGGACGAG ATTTTGCGTA CTCCACAGCT GCGTGAACTG TGGAAATCAG AACTGAATCA AATGAGTTCT CGCTTACAGG ACGTCCGCCA CCGTCTCGTT GCGCAGCTTC GTGCCTGTGG CTCTACCCGC GATTGGGAGT ACCTGGAAAA AGGCGTCGGC ATGATGTCCC TCACGGGTCT GACTGAAGAG CAAGTAATGG CGCTGCAGCA AAAATATTCC GTGTATTTGA CGCGTAATGG CCGTATCGCT TTTAGTGGCT TAAGTTCAGA AAATGTAGCA TATGTAGCGC AGTCTATCCA TGATGTTTCC CGCTAA
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **


 * *GC% Content for gene (codon optimized): ** 53.94%

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

**
 * Primer design results for 'tail' primers (this is just 2 sequences): **