Target+- FabI+enoyl-acyl-carrier-protein+reductase+NADH+(Wolbachia)


 * Target (protein/gene name):** FabI enoyl-[acyl-carrier-protein] reductase NADH

07/29/13

Primers needed to clone into pET-27b(+) vector. Cut sites will be EcoRI and HindIII

Forward Primer:

ATCTATACTTGAATTCGATGGCGATTTCTCTGCT

Reverse Primer (Reverse Complemented):

GTAACCGGCTAAAAGCTTTCAATTTTGGTAAGA

Notes: 1. Temp down 2. Truncate 3. Clone into a pET vector

Adding detergents, high salt concentrations, etc. didn't necessarily work when added to normal lysis buffer in preventing aggregation of FabI protein. It should be noted that the protein actually became soluble with these added conditions more than normal conditions.

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102712 GST tag info: @http://cshprotocols.cshlp.org/content/2007/4/pdb.prot4738.full

GST with His6 tag. LIC cloning (similar but different to our LIC cloning) @http://www.addgene.org/29655/ @http://qb3.berkeley.edu/qb3/macrolab/projects.cfm

From SGC - but requires special cloning system... @http://www.addgene.org/26101/ @http://www.sgc.utoronto.ca/SGC-WebPages/toronto-vectors.php


 * NCBI Gene # or RefSeq#:** 3266932

MHS Rank: 114 GCS Rank: 137 on WOLTR spreadsheet
 * Protein ID (NP or XP #) or Wolbachia#:** __**Protein Accession #**__ : AAW71058.1 __**Wolbachia #**__ : Q5GSG6 **__Locus:__**YP_198300


 * Organism:** Wolbachia endosymbiont strain TRS of Brugia malayi


 * Etiologic Risk Group (see link below):** N/A

29360.4 kDa
 * Molecular Weight:**

Wolbachia are gram-negative bacteria that form intracellular inherited infections in many invertebrates. They are extremely common with 20-75% of all insects being infected. Moreover they infect numerous non-insect invertebrates including nematodes, mites and spiders. The limits of the host range of Wolbachia are not fully appreciated at this time. Much of the success of Wolbachia can be attributed to the diverse phenotypes that result from infection. These range from classical mutualism to reproductive parasitism as characterized by the ability of Wolbachia to override chromosomal sex determination, induce parthenogenesis, selectively kill males, influence sperm competition and generate cytoplasmic incompatibility in early embryos.
 * Background/Disease Information:**

from: https://www.wolbachiawebsite.org/about.cfm

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 * Essentiality of this protein: Found to be essential in E. Coli lipid synthesis**


 * Complex of enzymes:** No


 * EC#:** 1.3.1.9


 * Link to BRENDA EC# page:** http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.3.1.9


 * **Reaction catalyzed by enoyl-[acyl-carrier-protein] reductase (NADH) (1.3.1.9)** ||  ||




 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):**
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates):** see below
 * -- link to paper that contains assay information:**see below; full link---http://pubs.acs.org/doi/full/10.1021/jm020050%2B

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 * Structure Available (PDB or Homology model):**
 * -- PDB # or closest PDB entry if using homology model: 3K2E**
 * -- Query Coverage (if not direct match): 97%**
 * -- Max Ident (if not direct match): 65%**


 * Druggable Target (see Databases for this): Found to be druggable in E. Coli**


 * Current Inhibitors:**

code format="genbank" 1 maisllqgkk glitgiinkr siaygivktl sehgaesavt yqneivkerl lsiaaelnve 61 lvlncdvane gtiddvfksi eekwgtldfl vhaiafsdkn elsgkyvnts lnnfqnamni 121 scysftalaq raekmmpngg slltlsyyga ekvmpnynvm glckaaleas vkylacdlgp 181 qnirvnaisa gpirtlassg isdfhfisew nrnnsplrrn ttledvgkaa lyllsdlssg 241 ttgeilhvds gynvvgmkai dsniinsyqn code
 * Expression Information (has it been expressed in bacterial cells):**
 * Purification Method:**
 * Image of protein (PyMol or etc):**
 * Amino Acid Sequence:**


 * length of your protein in Amino Acids: 270**
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website**
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**
 * CDS Gene Sequence:**

ATGGCGATTTCTCTGCTGCAGGGCAAAAAAGGTCTGATCACCGGCATTATCAACAAACGT TCTATCGCGTACGGTATCGTTAAAACCCTGAGCGAACATGGTGCAGAATCTGCGGTTACC TACCAGAACGAAATCGTCAAAGAACGTCTGCTGAGCATTGCCGCGGAACTGAACGTTGAA CTGGTTCTGAACTGCGACGTTGCGAACGAAGGTACCATCGACGACGTTTTCAAATCTATC GAAGAAAAATGGGGTACGCTCGACTTTCTGGTTCACGCAATCGCATTCTCTGACAAAAAC GAACTGTCTGGTAAATACGTTAACACCTCTCTGAACAATTTCCAAAACGCCATGAACATC TCTTGCTACTCTTTCACTGCGCTGGCGCAACGCGCTGAAAAGATGATGCCAAATGGTGGT TCTCTCCTGACGCTGTCTTACTACGGTGCGGAAAAAGTTATGCCGAACTACAATGTTATG GGTCTGTGCAAAGCGGCTCTGGAAGCGTCTGTTAAATACCTGGCGTGCGACCTGGGTCCG CAGAACATCCGTGTAAACGCGATCTCTGCGGGTCCGATCCGTACCCTGGCGAGCTCTGGT ATTTCTGACTTCCACTTCATCAGCGAATGGAACCGTAACAACTCTCCGCTGCGTCGTAAC ACCACCCTGGAAGACGTTGGTAAAGCCGCCCTGTACCTGCTGTCTGACCTGTCCTCTGGC ACGACGGGTGAGATCCTGCACGTTGACTCTGGTTACAACGTAGTTGGTATGAAAGCGATC GATTCTAACATCATTAATTCTTACCAAAATTGA


 * GC% Content for gene:** 49.2%
 * CDS Gene Sequence (codon optimized) - after having done Primer Design:**

ATGGCGATTTCTCTGCTGCAGGGCAAAAAAGGTCTGATCACCGGCATTATCAACAAACGT TCTATCGCGTACGGTATCGTTAAAACCCTGAGCGAACATGGTGCAGAATCTGCGGTTACC TACCAGAACGAAATCGTCAAAGAACGTCTGCTGAGCATTGCCGCGGAACTGAACGTTGAA CTGGTTCTGAACTGCGACGTTGCGAACGAAGGTACCATCGACGACGTTTTCAAATCTATC GAAGAAAAATGGGGTACGCTCGACTTTCTGGTTCACGCAATCGCATTCTCTGACAAAAAC GAACTGTCTGGTAAATACGTTAACACCTCTCTGAACAATTTCCAAAACGCCATGAACATC TCTTGCTACTCTTTCACTGCGCTGGCGCAACGCGCTGAAAAGATGATGCCAAATGGTGGT TCTCTCCTGACGCTGTCTTACTACGGTGCGGAAAAAGTTATGCCGAACTACAATGTTATG GGTCTGTGCAAAGCGGCTCTGGAAGCGTCTGTTAAATACCTGGCGTGCGACCTGGGTCCG CAGAACATCCGTGTAAACGCGATCTCTGCGGGTCCGATCCGTACCCTGGCGAGCTCTGGT ATTTCTGACTTCCACTTCATCAGCGAATGGAACCGTAACAACTCTCCGCTGCGTCGTAAC ACCACCCTGGAAGACGTTGGTAAAGCCGCCCTGTACCTGCTGTCTGACCTGTCCTCTGGC ACGACGGGTGAGATCCTGCACGTTGACTCTGGTTACAACGTAGTTGGTATGAAAGCGATC GATTCTAACATCATTAATTCTTACCAAAATTGA


 * GC% Content for gene (codon optimized):** 49.2%

__**Amino Acid Sequence:**__

MAISLLQGKKGLITGIINKRSIAYGIVKTLSEHGAESAVTYQNEIVKERLLSIAAELNVE LVLNCDVANEGTIDDVFKSIEEKWGTLDFLVHAIAFSDKNELSGKYVNTSLNNFQNAMNI SCYSFTALAQRAEKMMPNGGSLLTLSYYGAEKVMPNYNVMGLCKAALEASVKYLACDLGP QNIRVNAISAGPIRTLASSGISDFHFISEWNRNNSPLRRNTTLEDVGKAALYLLSDLSSG TTGEILHVDSGYNVVGMKAIDSNIINSYQN


 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):**


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


 * __Upstream:__** TACTTCCAATCC ATG __GCGATTTCTCTGCTG__
 * __Downstream:__** TATCCACCTTTACTG TCA __ATTTTGGTAAGAATTAATG__

FabI used in fatty acid/ lipid biosynthesis

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Journal Article: []

0.25 mM crotonoyl-CoA: Product Number: 28007, 5 mg $246.00 50 μM NADH: Product Number: N4505, 100 mg $58.80
 * Substrates needed: From Sigma**

http://www.ncbi.nlm.nih.gov/protein/58419043

Q5GSG6

Protein Accession # AAW71058.1


 * Primer Link LogFile from DNA works Primer Design:** https://docs.google.com/open?id=0B4O2KqKh2q_-RGlfc3YyVnJ6ZFU

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 * Link to Primer Design** -

__**Plasmid (FABI) inserted into vector**__ TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGT AGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGCGATTTCTCTGCTGCAGGGCAA AAAAGGTCTGATCACCGGCATTATCAACAAACGTTCTATCGCGTACGGTATCGTTAAAAC CCTGAGCGAACATGGTGCAGAATCTGCGGTTACCTACCAGAACGAAATCGTCAAAGAACG TCTGCTGAGCATTGCCGCGGAACTGAACGTTGAACTGGTTCTGAACTGCGACGTTGCGAA CGAAGGTACCATCGACGACGTTTTCAAATCTATCGAAGAAAAATGGGGTACGCTCGACTT TCTGGTTCACGCAATCGCATTCTCTGACAAAAACGAACTGTCTGGTAAATACGTTAACAC CTCTCTGAACAATTTCCAAAACGCCATGAACATCTCTTGCTACTCTTTCACTGCGCTGGC GCAACGCGCTGAAAAGATGATGCCAAATGGTGGTTCTCTCCTGACGCTGTCTTACTACGG TGCGGAAAAAGTTATGCCGAACTACAATGTTATGGGTCTGTGCAAAGCGGCTCTGGAAGC GTCTGTTAAATACCTGGCGTGCGACCTGGGTCCGCAGAACATCCGTGTAAACGCGATCTC TGCGGGTCCGATCCGTACCCTGGCGAGCTCTGGTATTTCTGACTTCCACTTCATCAGCGA ATGGAACCGTAACAACTCTCCGCTGCGTCGTAACACCACCCTGGAAGACGTTGGTAAAGC CGCCCTGTACCTGCTGTCTGACCTGTCCTCTGGCACGACGGGTGAGATCCTGCACGTTGA CTCTGGTTACAACGTAGTTGGTATGAAAGCGATCGATTCTAACATCATTAATTCTTACCA AAATTGACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCC GCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAA GCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAA CGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACG CGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTA CACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGT TCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTG CTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCAT CGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGAC TCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAG GGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACG CGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGC GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATT AATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTA TCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAG TTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATA CAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTG ACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACA GGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGT GATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGA ATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCA GGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCAT GCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGC CAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTC AGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGC CCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAAT CGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTG TTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCT GCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACC AAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA TCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTG ATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGT GGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGA GCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTAC GCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGAT GCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGC CCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCG CTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCAT CACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCAC AGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCT GGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCT CCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGC TCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAAC AACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCT TCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCC GGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAAC CGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACG TTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCC GGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAA TGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGG CGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGC GGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAA AGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGA CTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAAC TTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTG GTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGA GAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATG GTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAG ATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATC TGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTT TGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGA TTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGT CGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCA AGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCC AGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCT TTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAG GTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGG CTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACA TCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTAT CATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCC CTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGC CGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCC TGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTC CCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCC GGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAAT

Homology model site: @http://www1.jcsg.org/scripts/prod/scwrl/serve.cgi

TMPred site: @http://www.ch.embnet.org/software/TMPRED_form.html