Glutamine+Synthetase

=***Target (protein/gene name):** Glutamine Synthetase GlnA1=


 * NCBI Gene #:** 888383


 * Protein ID (NP or XP #:** MTCY190.31, MTCY427.01


 * Organism (including strain):** //Mycobacterium tuberculosis// (strain ATCC 25618/ H37Rv)


 * Etiologic Risk Group (see link below):** Risk Group 2 (WHO)

//Mycobacterium tuberculosis// is the pathogen that causes tuberculosis, a virulent disease that has infected a large portion of the world's population. It is spread through aerosols, so actions such as coughing or sneezing from an infected individual may release the pathogen into the air. Once in the air, the pathogen may be taken into the body by others, increasing the spread of infection. Once the pathogen enters the body, it multiplies, causing a response from the immune system. This is why individuals who are immunodeficient are highly susceptible to tuberculosis. Though the pathogen normally targets the lungs, other parts of the body are susceptible too. The symptoms of this disease include chronic coughing, chest pain, general malaise, fever, chills, and even weight loss.
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**

http://tdrtargets.org/targets/view?gene_id=6148
 * Link to TDR Targets page (if present):**

http://www.ncbi.nlm.nih.gov/gene/888383
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):**

Glutamine synthetase is significant in metabolism of nitrogen within the pathogen. This enzyme catalyzes the condensation of glutamate and ammonia to form glutamine, one of the twenty amino acids. Without glutamine, synthesis of many proteins will be inhibited. Without a few of these proteins, the pathogen's viability will effectively reduced.
 * Essentiality of this protein:**

The essentiality data:


 * Gene/Ortholog:** mtu2255 (OG4_10454); **Phenotype:** essential; **Source study:** nmpdr
 * Gene/Ortholog:** mtu1908 (OG4_10454); **Phenotype:** non-essential; **Source study:** nmpdr
 * Gene/Ortholog:** mtu2907 (OG4_10454); **Phenotype:** non-essential; **Source study:** nmpdr
 * Gene/Ortholog:** mtu1909 (OG4_10454); **Phenotype:** non-essential; **Source study:** nmpdr
 * Gene/Ortholog:** eco3728 (OG4_10454); **Phenotype:** non-essential; **Source study:** blattner
 * Gene/Ortholog:** eco3728 (OG4_10454); **Phenotype:** non-essential; **Source study:** gerdes
 * Gene/Ortholog:** eco3728 (OG4_10454); **Phenotype:** non-essential; **Source study:** keio
 * Gene/Ortholog:** eco3728 (OG4_10454); **Phenotype:** non-essential; **Source study:** shigen


 * Complex of proteins?:** No

Escherichia coli: http://www.ncbi.nlm.nih.gov/pubmed/16190760
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):**


 * *EC#: ** 6.3.1.2

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=6.3.1.2
 * Link to BRENDA EC# page:**


 * --** Show screenshot of BRENDA enzyme mechanism schematic



Reagents: 100 mM Imidazole HCL buffer, pH 7.1 at 37 degrees Celsius, 3M Sodium Glutamate Solution, 250mM Adenosine 5' Triphosphate solution
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):** Continuous Spectrophotometric Rate Determination

http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number-v.html#-%206%20-
 * -- 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/glutaminesynthetase.pdf
 * -- links to assay reagents (substrates) pages.**

Imidazole- $17.10, 1 gram, Sigma-Aldrich, I-0250 L-Glutamic acid monosodium salt monohydrate- $34.50, 250 grams, Sigma-Aldrich, 49621 Adenosine 5'-triphosphate disodium salt hydrate- $46.90, 1 gram, Sigma-Aldrich, A2383
 * --- List cost and quantity of substrate reagents, supplier, and catalog #**

-- PDB #: 1HTO
 * Structure Available (PDB or Homology model)**


 * Current Inhibitors:** Adenosine Monophosphate, Citric Acid, Manganese (II) Ion


 * Expression Information (has it been expressed in bacterial cells):** Expression System was Escherichia coli

E. coli cells are used to produce the glutamine synthetase. The cells then undergo centrifugation using GSA rotor at 4 degrees Celsius for 15 minutes. The pellets are suspended in 10mM of potassium phosphate/10mM 2-mercaptoethanol and centrifuged again. The cells are re-suspended in 10mL of phosphate buffer per gram of cells and put on ice for 10 minutes. Centrifugation follows. The supernatant is then loaded onto a hydroxyapatite column equilibrated in 10mM potassium phosphate/10mM 2-mercaptoethanol. The supernatant is eluted and the elution is centrifuged to collect the purified protein.
 * Purification Method:**

code MTEKTPDDVFKLAKDEKVEYVDVRFCDLPGIMQHFTIPASAFDKSVFDDGLAFDGSSIRG FQSIHESDMLLLPDPETARIDPFRAAKTLNINFFVHDPFTLEPYSRDPRNIARKAENYLI STGIADTAYFGAEAEFYIFDSVSFDSRANGSFYEVDAISGWWNTGAATEADGSPNRGYKV RHKGGYFPVAPNDQYVDLRDKMLTNLINSGFILEKGHHEVGSGGQAEINYQFNSLLHAAD DMQLYKYIIKNTAWQNGKTVTFMPKPLFGDNGSGMHCHQSLWKDGAPLMYDETGYAGLSD TARHYIGGLLHHAPSLLAFTNPTVNSYKRLVPGYEAPINLVYSQRNRSACVRIPITGSNP KAKRLEFRSPDSSGNPYLAFSAMLMAGLDGIKNKIEPQAPVDKDLYELPPEEAASIPQTP TQLSDVIDRLEADHEYLTEGGVFTNDLIETWISFKRENEIEPVNIRPHPYEFALYYDV code
 * Image of protein (PyMol with features delineated and shown separately):**
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code')**
 * length of your protein in Amino Acids:** 478


 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website:** 53.57 kDa


 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 63385 M^-1 cm^-1


 * TMpred graph Image **

code format="genbank" 1 gtgacggaaa agacgcccga cgacgtcttc aaacttgcca aggacgagaa ggtcgaatat 61 gtcgacgtcc ggttctgtga cctgcctggc atcatgcagc acttcacgat tccggcttcg 121 gcctttgaca agagcgtgtt tgacgacggc ttggcctttg acggctcgtc gattcgcggg 181 ttccagtcga tccacgaatc cgacatgttg cttcttcccg atcccgagac ggcgcgcatc 241 gacccgttcc gcgcggccaa gacgctgaat atcaacttct ttgtgcacga cccgttcacc 301 ctggagccgt actcccgcga cccgcgcaac atcgcccgca aggccgagaa ctacctgatc 361 agcactggca tcgccgacac cgcatacttc ggcgccgagg ccgagttcta cattttcgat 421 tcggtgagct tcgactcgcg cgccaacggc tccttctacg aggtggacgc catctcgggg 481 tggtggaaca ccggcgcggc gaccgaggcc gacggcagtc ccaaccgggg ctacaaggtc 541 cgccacaagg gcgggtattt cccagtggcc cccaacgacc aatacgtcga cctgcgcgac 601 aagatgctga ccaacctgat caactccggc ttcatcctgg agaagggcca ccacgaggtg 661 ggcagcggcg gacaggccga gatcaactac cagttcaatt cgctgctgca cgccgccgac 721 gacatgcagt tgtacaagta catcatcaag aacaccgcct ggcagaacgg caaaacggtc 781 acgttcatgc ccaagccgct gttcggcgac aacgggtccg gcatgcactg tcatcagtcg 841 ctgtggaagg acggggcccc gctgatgtac gacgagacgg gttatgccgg tctgtcggac 901 acggcccgtc attacatcgg cggcctgtta caccacgcgc cgtcgctgct ggccttcacc 961 aacccgacgg tgaactccta caagcggctg gttcccggtt acgaggcccc gatcaacctg 1021 gtctatagcc agcgcaaccg gtcggcatgc gtgcgcatcc cgatcaccgg cagcaacccg 1081 aaggccaagc ggctggagtt ccgaagcccc gactcgtcgg gcaacccgta tctggcgttc 1141 tcggccatgc tgatggcagg cctggacggt atcaagaaca agatcgagcc gcaggcgccc 1201 gtcgacaagg atctctacga gctgccgccg gaagaggccg cgagtatccc gcagactccg 1261 acccagctgt cagatgtgat cgaccgtctc gaggccgacc acgaatacct caccgaagga 1321 ggggtgttca caaacgacct gatcgagacg tggatcagtt tcaagcgcga aaacgagatc 1381 gagccggtca acatccggcc gcatccctac gaattcgcgc tgtactacga cgtttaa code
 * CDS Gene Sequence (paste as text only):**
 * GC% Content for gene:** 61.238%

GTGACCGAAA AGACCCCGGA CGATGTTTTC AAACTGGCGA AAGACGAAAA AGTGGAATAC GTGGATGTGC GCTTCTGTGA TCTGCCGGGT ATTATGCAAC ACTTTACAAT TCCTGCGTCG GCCTTTGATA AATCCGTCTT TGACGACGGC CTTGCATTTG ATGGCAGCTC CATCCGGGGA TTTCAAAGTA TTCATGAATC CGACATGCTG TTACTGCCGG ATCCGGAAAC AGCTCGTATT GATCCCTTTC GTGCGGCAAA AACCCTGAAT ATTAATTTCT TTGTGCATGA TCCTTTCACC CTGGAACCGT ACAGCCGCGA TCCTCGCAAC ATCGCCCGTA AAGCGGAAAA CTATCTGATC TCAACTGGAA TTGCTGATAC CGCGTACTTT GGCGCAGAGG CCGAGTTCTA CATTTTTGAC AGCGTATCTT TCGACTCTCG CGCGAACGGC AGCTTTTACG AAGTGGATGC AATCTCGGGG TGGTGGAACA CCGGGGCCGC TACCGAAGCT GATGGCTCGC CAAATCGCGG CTACAAGGTA CGCCACAAGG GCGGTTACTT TCCTGTCGCT CCAAACGACC AGTACGTGGA TCTGCGTGAC AAGATGCTCA CCAACCTGAT TAATTCCGGC TTTATTCTGG AAAAAGGCCA TCATGAGGTT GGGAGTGGGG GTCAGGCAGA AATTAACTAC CAATTTAACT CCCTGTTACA TGCTGCGGAC GACATGCAAC TGTACAAATA CATCATCAAA AACACGGCTT GGCAAAACGG TAAAACGGTG ACATTTATGC CGAAGCCACT GTTTGGAGAC AATGGCTCTG GCATGCATTG TCACCAATCG CTTTGGAAAG ATGGAGCGCC TCTGATGTAT GATGAAACCG GCTATGCTGG GCTGAGCGAT ACGGCCCGCC ACTATATTGG TGGGTTACTG CACCATGCGC CATCACTGCT GGCTTTTACG AATCCGACCG TTAATAGTTA TAAACGCTTG GTTCCGGGAT ACGAAGCGCC AATTAACCTG GTGTATAGCC AGCGTAACCG CTCCGCTTGC GTTCGTATCC CGATCACCGG CTCTAATCCA AAAGCAAAAC GTTTAGAGTT CCGTTCGCCC GACAGTAGTG GTAACCCTTA TTTGGCGTTT AGTGCGATGC TGATGGCAGG CTTGGACGGC ATTAAAAATA AAATCGAGCC TCAGGCACCG GTCGATAAAG ATTTATATGA ACTTCCGCCC GAAGAGGCGG CATCCATCCC GCAGACTCCG ACGCAACTGT CGGATGTAAT TGATCGCCTG GAGGCCGATC ATGAGTACCT GACGGAGGGG GGTGTCTTCA CGAACGATCT GATTGAAACC TGGATCTCGT TCAAACGTGA AAACGAAATT GAACCTGTTA ATATTCGCCC GCACCCTTAT GAATTTGCCC TGTATTATGA TGTGTAA
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**


 * GC% Content for gene (codon optimized):** 49.756%

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):**