Zinc+metalloproteinase, Bacillus+anthracis

https://www.ncbi.nlm.nih.gov/gene/3361711 Is it a monomer or multimer as biological unit ** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): monomer with 4 domains https://www.rcsb.org/structure/4PKT Maize, K.; Kurbanov, E.; De La Mora-Rey, T.; Geders, T.; Hwang, D.-J.; Walters, M.; Johnson, R.; Amin, E.; Finzel, B., Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding. //Acta Crystallogr D Biol Crystallogr// **2014,** //70// (11), 2813-2822.
 * *Target (protein/gene name): ** Zinc metalloproteinase (also called lethal factor endopeptidase)
 * *NCBI Gene # or RefSeq#: ** 3361711 (NCBI #)
 * *Protein ID (NP or XP #) or Wolbachia#: ** 3.4.24.83
 * *Organism (including strain): ** //Bacillus anthracis//
 * Etiologic Risk Group (see link below): ** Level 4 risk group (category A priority pathogen)
 * */ Disease Information (sort of like the Intro to your Mini Research Write up): ** //Bacillus anthracis// is a bacteria that can cause infection in the lungs, the intestinal tract, in the blood by injection, or topically on the skin. Even with treatment, inhalation of anthrax has between a 50 and 80% mortality rate. Due to its virulence it has been developed into a weapon by many countries and a treatment for it is high priority since it has so much potential as a bioterrorist weapon. The toxin that anthrax secretes is made of three parts - the lethal factor, edema factor, and protective antigen. The toxin can have continued effects in a persons body even after the bacteria itself has been killed which is why a treatment for the toxin is needed as well as antibiotics.
 * Link to TDR Targets page (if present): ** n/a
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): **
 * Essentiality of this protein: ** Essential for the virulence of the organism in the toxin that it secretes
 * Complex of proteins?: ** no
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): ** evidence of binding in PDB entries

http://www.brenda-enzymes.org/enzyme.php?ecno=3.4.24.83 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805240/ https://www.sigmaaldrich.com/catalog/substance/dimethylsulfoxide78136768511?lang=en®ion=US&attrlist=Assay https://www.listlabs.com/products/buy-mapkkide-peptide-substrate-o-abzdnpfor-anthrax-lethal-factor
 * *EC#: ** 3 (hydrolase, 3.4.24.83)
 * Link to BRENDA EC# page: **
 * -- Show screenshot of BRENDA enzyme mechanism schematic **
 * 83 || kDa full-length protective antigen || + ||  || H2O || = || 20 || kDa N-terminal fragment of protective antigen || + || 63 || kDa N-terminal fragment of protective antigen ||
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): ** FRET (Forster Resonance Energy Transfer) with a DMSO solution and Abz/Dnp anthrax lethal factor substrate
 * -- -or link (or citation) to paper that contains assay information **
 * -- links to assay reagents (substrates) pages. **


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

DMSO: $40.50 (50 mL), Sigma Aldrich, D8418

Abz/DNP-substrate: $285.00 (200 nmoles), List Biological Laboratories, Inc., 530

-- PDB # or closest PDB entry if using homology model: 4PKT
 * Structure (PDB or Homology model) **

SNALSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKL
 * Current Inhibitors: ** listed on BRENDA and DB binding
 * Expression Information (has it been expressed in bacterial cells): ** E. coli BL21 (DE3)
 * Purification Method : ** Ni-NTA column and Hiprep 26/60 Sephacryl S-200 HR column
 * Image of protein (PyMol with features delineated and shown separately): **
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **

QIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDA

LLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKW

RIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTK

LITFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEIYEQVHSKGLYVP

ESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAF

RLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINSLVPR ATGAATATAAAAAAAGAATTTATAAAAGTAATTAGTATGTCATGTTTAGTAACAGCAATTACTTTGAGTG GTCCCGTCTTTATCCCCCTTGTACAGGGGGCGGGCGGTCATGGTGATGTAGGTATGCACGTAAAAGAGAA AGAGAAAAATAAAGATGAGAATAAGAGAAAAGATGAAGAACGAAATAAAACACAGGAAGAGCATTTAAAG GAAATCATGAAACACATTGTAAAAATAGAAGTAAAAGGGGAGGAAGCTGTTAAAAAAGAGGCAGCAGAAA AGCTACTTGAGAAAGTACCATCTGATGTTTTAGAGATGTATAAAGCAATTGGAGGAAAGATATATATTGT GGATGGTGATATTACAAAACATATATCTTTAGAAGCATTATCTGAAGATAAGAAAAAAATAAAAGACATT TATGGGAAAGATGCTTTATTACATGAACATTATGTATATGCAAAAGAAGGATATGAACCCGTACTTGTAA TCCAATCTTCGGAAGATTATGTAGAAAATACTGAAAAGGCACTGAACGTTTATTATGAAATAGGTAAGAT ATTATCAAGGGATATTTTAAGTAAAATTAATCAACCATATCAGAAATTTTTAGATGTATTAAATACCATT AAAAATGCATCTGATTCAGATGGACAAGATCTTTTATTTACTAATCAGCTTAAGGAACATCCCACAGACT TTTCTGTAGAATTCTTGGAACAAAATAGCAATGAGGTACAAGAAGTATTTGCGAAAGCTTTTGCATATTA TATCGAGCCACAGCATCGTGATGTTTTACAGCTTTATGCACCGGAAGCTTTTAATTACATGGATAAATTT AACGAACAAGAAATAAATCTATCCTTGGAAGAACTTAAAGATCAACGGATGCTGTCAAGATATGAAAAAT GGGAAAAGATAAAACAGCACTATCAACACTGGAGCGATTCTTTATCTGAAGAAGGAAGAGGACTTTTAAA AAAGCTGCAGATTCCTATTGAGCCAAAGAAAGATGACATAATTCATTCTTTATCTCAAGAAGAAAAAGAG CTTCTAAAAAGAATACAAATTGATAGTAGTGATTTTTTATCTACTGAGGAAAAAGAGTTTTTAAAAAAGC <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">TACAAATTGATATTCGTGATTCTTTATCTGAAGAAGAAAAAGAGCTTTTAAATAGAATACAGGTGGATAG <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">TAGTAATCCTTTATCTGAAAAAGAAAAAGAGTTTTTAAAAAAGCTGAAACTTGATATTCAACCATATGAT <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">ATTAATCAAAGGTTGCAAGATACAGGAGGGTTAATTGATAGTCCGTCAATTAATCTTGATGTAAGAAAGC <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">AGTATAAAAGGGATATTCAAAATATTGATGCTTTATTACATCAATCCATTGGAAGTACCTTGTACAATAA <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">AATTTATTTGTATGAAAATATGAATATCAATAACCTTACAGCAACCCTAGGTGCGGATTTAGTTGATTCC <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">ACTGATAATACTAAAATTAATAGAGGTATTTTCAATGAATTCAAAAAAAATTTCAAATATAGTATTTCTA <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">GTAACTATATGATTGTTGATATAAATGAAAGGCCTGCATTAGATAATGAGCGTTTGAAATGGAGAATCCA <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">ATTATCACCAGATACTCGAGCAGGATATTTAGAAAATGGAAAGCTTATATTACAAAGAAACATCGGTCTG <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">GAAATAAAGGATGTACAAATAATTAAGCAATCCGAAAAAGAATATATAAGGATTGATGCGAAAGTAGTGC <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">CAAAGAGTAAAATAGATACAAAAATTCAAGAAGCACAGTTAAATATAAATCAGGAATGGAATAAAGCATT <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">AGGGTTACCAAAATATACAAAGCTTATTACATTCAACGTGCATAATAGATATGCATCCAATATTGTAGAA <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">AGTGCTTATTTAATATTGAATGAATGGAAAAATAATATTCAAAGTGATCTTATAAAAAAGGTAACAAATT <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">ACTTAGTTGATGGTAATGGAAGATTTGTTTTTACCGATATTACTCTCCCTAATATAGCTGAACAATATAC <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">ACATCAAGATGAGATATATGAGCAAGTTCATTCAAAAGGGTTATATGTTCCAGAATCCCGTTCTATATTA <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">CTCCATGGACCTTCAAAAGGTGTAGAATTAAGGAATGATAGTGAGGGTTTTATACACGAATTTGGACATG <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">CTGTGGATGATTATGCTGGATATCTATTAGATAAGAACCAATCTGATTTAGTTACAAATTCTAAAAAATT <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">CATTGATATTTTTAAGGAAGAAGGGAGTAATTTAACTTCGTATGGGAGAACAAATGAAGCGGAATTTTTT <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">GCAGAAGCCTTTAGGTTAATGCATTCTACGGACCATGCTGAACGTTTAAAAGTTCAAAAAAATGCTCCGA <span style="font-family: &#39;Courier New&#39;; font-size: 8pt;">AAACTTTCCAATTTATTAACGATCAGATTAAGTTCATTATTAACTCATAA
 * *length of your protein in Amino Acids: ** 519 amino acids
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: ** 60.35126 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 58790
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * *CDS Gene Sequence (paste as text only): **
 * <span style="font-family: monospace,serif;">*GC% Content for gene: **30%
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **
 * *GC% Content for gene (codon optimized): **

-- 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): **