GMP+Synthase+Methanocaldococcus+jannaschii


 * *Target (protein/gene name): ****GMP Synthase **

 ***NCBI Gene # or RefSeq#:** WP_010871100.1

 ***Organism (including strain):** ** Methanocaldococcus jannaschii **

 **Etiologic Risk Group (see link below):** **n/a **


 * */ ** **Disease Information ** **(sort of like the Intro to your Mini ****Research Write ****up): **
 * Methanocaldococcus jannaschii **** is part of the archaea domain and are single celled organisms. They are found in extreme environments such as the hypothermal vents at the bottom of the ocean where water boils and has high pressure. This organism has been found to have the same efflux pumps and permeases that transport hydrophobic drugs out of certain bacteria causing the bacteria not to be affected. If they have these same pumps/permeases, then M. jannaschii would be able to be unaffected by those drugs and cause harm to humans if the organism invaded. **

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

 **Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):** **https://www.ncbi.nlm.nih.gov/protein/WP_010871100.1 **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Essentiality of this protein:** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">GMP synthase is used to synthesize GMP from XMP which is a part of purine metabolism. **

<span style="background-color: #ffffff; font-family: Arial,sans-serif; font-size: 10pt;">Is it a monomer or multimer as biological unit **<span style="font-family: Arial,sans-serif; font-size: 10pt;">? (make prediction at ** <span style="font-family: Arial,sans-serif; font-size: 10pt;">@http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html <span style="background-color: #ffffff; font-family: Arial,sans-serif; font-size: 10pt;">): <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">Monomer

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Complex of proteins?:** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">no **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">not available **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***EC#:** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">6. 3. 5. 2 **


 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Link to BRENDA EC# page: ****<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.5.2 **

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

<span class="m9l40l772" 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: Arial,sans-serif; font-size: 10pt;"> **-- link to Sigma (or other** <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="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/b6916bul.pdf

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **-- -or link (or citation) to paper that contains assay information** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859818/#R38 **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **-- links to assay reagents (substrates) pages.** <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">https://www.sigmaaldrich.com/catalog/product/sigma/b6916?lang=en®ion=US


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

<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">GMPS can be assayed via dye binding with bovine serum albumin standard and a spectrophotometer. Bradford reagent contains Brilliant Blue G in phosphoric acid and methanol. To assay, you mix one part protein to thirty part reagent and run it through the spectrophotometer and recorded at absorbance of 595 nm. The Sigma Aldrich catalog number for the reagent is B6916 and it costs $70.00 for 500 ml.


 * <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: <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">2LXN

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Current Inhibitors:** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">none directly for Methanocaldococcus jannaschii **<span style="font-family: Arial,sans-serif; font-size: 10pt;">. For expression in //<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">E. coli //<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">, inhibitors include 2-fluoroinosine 5’-monophosphate, 1-methyladenosine, and 2’-deoxyadenosine.

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Expression Information (has it been expressed in bacterial cells):** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">expressed in //E. coli// **

<span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Purification Method** <span style="font-family: Arial,sans-serif; font-size: 10pt;">**:** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">GMP synthase can be purified by having a 4M solution of the protein denaturant guanidinium thiocyanate and .1 M 2-mercaptoethanol to break the disulfide bonds. The GMP can be filtered by ethanol precipitation or sedimentation with cesium chloride. **

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


 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">Figure 1. Structure of 2LXN from Methanocaldococcus jannaschii **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):** <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">MIVILDNGGQYVHRIHRSLKYIGVSSKIVPNTTPLEEIESNKEVKGIILSGGPDIEKAKN <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">CIDIALNAKLPILGICLGHQLIALAYGGEVGRAEAEEYALTKVYVDKENDLFKNVPREFN <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">AWASHKDEVKKVPEGFEILAHSDICQVEAMKHKTKPIYGVQFHPEVAHTEYGNEILKNFC <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">KVCGYKFE

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***length of your protein in Amino Acids:** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">188 **

<span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Molecular Weight** <span style="font-family: Arial,sans-serif; font-size: 10pt;">**of your protein in kiloDaltons using the** [|**Expasy ProtParam**]**<span style="font-family: Arial,sans-serif; font-size: 10pt;">website: ****<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">21020 **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> **Molar** <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Extinction coefficient** <span style="font-family: Arial,sans-serif; font-size: 10pt;">**of your protein at 280 nm wavelength:** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">17420 **

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

Figure 2. TMpred output for 2LXN in **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">Methanocaldococcus jannaschii **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***CDS** <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Gene Sequence** <span style="font-family: Arial,sans-serif; font-size: 10pt;">**(paste as text only):** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">ATGATTGTTATCTTAGACAACGGAGGGCAGTATGTTCATAGAATACACAGAAGTTTAAAG **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">TATATTGGAGTTAGCTCAAAGATAGTTCCAAATACAACACCATTGGAAGAGATTGAAAGC **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">AATAAAGAAGTTAAGGGTATTATACTAAGTGGAGGGCCTGATATTGAAAAAGCTAAAAAT **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">TGTATAGATATTGCTTTAAATGCTAAACTCCCTATTTTGGGGATTTGTTTAGGACATCAA **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">TTAATTGCCTTGGCTTATGGTGGAGAGGTTGGAAGGGCTGAGGCAGAAGAATACGCACTA **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">ACAAAGGTCTATGTAGATAAAGAAAACGATTTATTTAAAAACGTTCCAAGAGAGTTCAAT **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">GCATGGGCTTCACATAAGGATGAGGTTAAAAAAGTTCCAGAAGGTTTTGAGATTTTAGCT **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">CATTCAGATATATGTCAGGTTGAAGCAATGAAACATAAAACAAAGCCGATTTATGGAGTT **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">CAGTTCCACCCTGAAGTTGCTCATACAGAATATGGAAATGAGATTTTGAAAAACTTTTGT **
 * <span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">AAAGTTTGTGGTTACAAATTTGAATAA **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***GC% Content for gene:** **<span style="color: #0070c0; font-family: Arial,sans-serif; font-size: 10pt;">34.39% **

<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ***CDS Gene Sequence (codon optimized) - copy from** <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**output** <span style="font-family: Arial,sans-serif; font-size: 10pt;">**of** <span class="m9l40l772" style="font-family: Arial,sans-serif; font-size: 10pt;">**Primer Design** <span style="font-family: Arial,sans-serif; font-size: 10pt;">**Protocol (paste as text only): n/a**

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