Target+-+Alpha+Carbonic+Anhydrase+(Helicobacter+pylori)

NP #: NC_000915 @http://www.ncbi.nlm.nih.gov/gene/899948 (use **Summer13E** Plate for Oligos instead of ordering new ones - Dr. B 090313)) - Used Summer13E Plate for Oligos - 091313
 * Target (protein/gene name):** Alpha Carbonate Dehydratase
 * NCBI Gene # or RefSeq#:** HPHPH27_0808 Change this - DR. B new gene iD: 899948 (9/13/13)
 * Protein ID (NP or XP #) or Wolbachia#:** EJB55340 - Get the WP # for this here. - Dr. B090213

//Helicobacter pylori // is a spiral-shaped bacterium commonly found in the stomach where the shape of the bacteria allow them to penetrate the stomach's protective mucous lining, where substances produced weaken the lining and make the stomach more susceptible to damage from gastric acids. In fact, most people infected with the bacteria never have symptoms or problems such as ulcers. Only a small number of people with the infection develop stomach cancer. But the m ost common symptom of peptic ulcer disease is gnawing or burning abdominal pain, usually in the area just beneath the ribs.  From research, it seems to be that the  frequency of people infected may somehow be related to race. About 60% of Hispanics and about 54% of African Americans have detectable organisms as compared to about 20%-29% of Anglo Americans. It is also known that children are very commonly infected in developing countries.
 * Organism (including strain):** //Helicobacter pylori//
 * Etiologic Risk Group:** Group I—Pathogens Newly Recognized in the Past Two Decades
 * Background/Disease Information:**

[]
 * Essentiality of this protein:** According to the article found, this protein is essential because carbonic anhydrases contributes to acid acclimation.
 * Complex of proteins?:**


 * *EC#: 4.2.1.1 **
 * Link to BRENDA EC# page:** []

@http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-carbonic-anhydrase.html
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): All information contained in this link.**
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates)**
 * -- link (or citation) to paper that contains assay information**
 * -- List cost and quantity of substrate reagents and supplier**

-- PDB # or closest PDB entry if using homology model: 1KOP (for homology model) -- For Homology Model option: Show pairwise alignment of your BLASTP search in NCBI against the PDB Query Coverage: 89% Max % Identities: 38% % Positives: 50% Chain used for homology:
 * Structure Available (PDB or Homology model)**

According to the article, acetazolamide and methazolamide were shown to inhibit the bacterial growth in vitro. @http://www.ncbi.nlm.nih.gov/pubmed/18336307 The protein appears to be toxic to E. coli, so a modified form of the gene lacking a part was prepared that encodes a cleavable signal peptide. [] In this article, the H. pylori enzyme was purified by sulfonamide affinity chromatography. [] no structure for this protein. MKKTFLIALALTASLIGAENTKWDYKNKENGPHRWDKLHKDFEV CKSGKSQSPINIEHYYHTQDKADLQFKYAASKPKAVFFTHHTLKASFEPTNHINYRGH DYVLDNVHFHAPMEFLINNKTRPLSAHFVHKDAKGRLLVLAIGFEEGKENPNLDPILE SVQKKQNFKEVALDAFLPKSINYYHFNGSLTAPPCTEGVAWFVIEEPLEVSAKQLAEI KKRMKNSPNQRPVQPDYNTVIIKSSAETR 28.3153 kDa Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 30035 Abs 0.1% (=1 g/l) 1.061, assuming all pairs of Cys residues form cystines Ext. coefficient 29910 Abs 0.1% (=1 g/l) 1.056, assuming all Cys residues are reduced atgaaaaaaacttttttgatcgctttagcacttacggcttctcttataggcgctgaaaataccaaatgggattataaaaataaaga aaatggcccgcaccgttgggacaaattgcacaaagattttgaagtgtgcaaaagcggtaaaagccaatcgcccatcaacatt gagcattactaccacacgcaagataaagccgatttgcaattcaaatacgccgcttccaaacctaaagcggtctttttcacccat cacactttaaaggcttcgtttgagccgactaaccacatcaattatagagggcatgactatgtgctggataatgtgcatttccacg cccctatggagtttttaatcaataataaaaccaggcctttgagcgcgcatttcgtgcataaagacgctaaagggcgtttgttagt gttagcgattggttttgaagaagggaaagaaaaccccaaccttgatcctattttagaaagcgttcaaaagaaacaaaattttaa agaggtggctttagacgctttcttgcctaaaagcatcaattactaccattttaacggctctctcaccgctcctccttgcacagaggg ggtggcatggtttgtcatagaagagcctttggaagtctctgccaaacaattggctgaaatcaaaaaacgcatgaaaaattcgcctaa ccaacgccccgtccagcctgactacaacaccgtgatcattaaaagctcagctgagacccgctaa
 * Current Inhibitors:**
 * Expression Information (has it been expressed in bacterial cells):** Based off the article,
 * Purification Method:**
 * 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:** 247 aa
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website**
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html).
 * CDS Gene Sequence (paste as text only):**
 * GC% Content for gene:** 42.3%
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol:**

SEQUENCE 1: PROTEIN LENGTH = 248



1 MKKTFLIALVLATSLIGAENAKWDYKNKENGPHRWDKLHKDFEVCKSGKSQSPINIEHYY

61 HTQDKADLQFKYAASKPKAVFFTHHTLKASFEPTNHINYRGHDYVLDNVHFHAPMEFLIN

121 NKTRPLSAHFVHKDAKGRLLVLAIGFEEGKENPNLDPILEGIQKKQNFKEVALDAFLPKS

181 INYYHFNGSLTAPPCTEGVAWFVIEEPLEVSAKQLAEIKKRMKNSPNQRPVQPDYNTVII

241 KSSAKTRX

1 ATGAAAAAAACCTTCCTGATCGCGCTGGTGCTGGCGACCTCTCTGATCGGTGCGGAGAAC 61 GCCAAATGGGACTACAAAAACAAAGAGAATGGTCCTCACCGTTGGGACAAACTGCACAAA 121 GACTTTGAAGTATGCAAGTCTGGTAAATCTCAGTCTCCGATCAACATCGAACACTACTAT 181 CACACCCAGGACAAAGCGGACCTGCAGTTCAAATACGCGGCGTCTAAACCGAAAGCGGTT 241 TTCTTCACCCACCACACCCTGAAAGCGTCTTTCGAACCGACCAACCACATCAACTACCGT 301 GGTCACGACTACGTTCTGGACAACGTTCACTTCCACGCGCCGATGGAATTTCTGATTAAC 361 AACAAAACCCGTCCGCTGTCTGCGCACTTCGTTCACAAGGACGCGAAAGGTCGTCTGCTG 421 GTTCTGGCAATCGGTTTCGAAGAAGGTAAAGAAAATCCTAACCTGGACCCGATCCTGGAA 481 GGTATCCAAAAGAAACAGAACTTTAAGGAAGTTGCGCTGGATGCGTTTCTCCCGAAATCT 541 ATCAATTACTACCACTTCAACGGCTCTCTCACGGCCCCTCCTTGCACCGAAGGTGTTGCG 601 TGGTTTGTTATCGAAGAGCCACTGGAGGTCTCTGCGAAACAGCTGGCGGAAATCAAAAAA 661 CGTATGAAGAACTCTCCGAACCAACGCCCGGTGCAGCCGGATTATAACACCGTTATCATC 721 AAATCTTCTGCAAAGACCCGTTAA

18 oligonucleotides need to be synthesized 1 ATGAAAAAAACCTTCCTGATCGCGCTGGTGCTGGCGACCTCTCTGATCGGTGCGGA 56 2 TGAGGACCATTCTCTTTGTTTTTGTAGTCCCATTTGGCGTTCTCCGCACCGATCAGAGAG 60 3 AAAAACAAAGAGAATGGTCCTCACCGTTGGGACAAACTGCACAAAGACTTTGAAGTATGC 60 4 GTTCGATGTTGATCGGAGACTGAGATTTACCAGACTTGCATACTTCAAAGTCTTTGTGCA 60 5 AGTCTCCGATCAACATCGAACACTACTATCACACCCAGGACAAAGCGGACCTGCAGTTCA 60 6 TGGTGGGTGAAGAAAACCGCTTTCGGTTTAGACGCCGCGTATTTGAACTGCAGGTCCGCT 60 7 CGGTTTTCTTCACCCACCACACCCTGAAAGCGTCTTTCGAACCGACCAACCACATCAACT 60 8 GTGGAAGTGAACGTTGTCCAGAACGTAGTCGTGACCACGGTAGTTGATGTGGTTGGTCGG 60 9 TGGACAACGTTCACTTCCACGCGCCGATGGAATTTCTGATTAACAACAAAACCCGTCCGC 60 10 GCAGACGACCTTTCGCGTCCTTGTGAACGAAGTGCGCAGACAGCGGACGGGTTTTGTTGT 60 11 CGCGAAAGGTCGTCTGCTGGTTCTGGCAATCGGTTTCGAAGAAGGTAAAGAAAATCCTAA 60 12 GTTTCTTTTGGATACCTTCCAGGATCGGGTCCAGGTTAGGATTTTCTTTACCTTCTTCGA 60 13 CCTGGAAGGTATCCAAAAGAAACAGAACTTTAAGGAAGTTGCGCTGGATGCGTTTCTCCC 60 14 GCCGTGAGAGAGCCGTTGAAGTGGTAGTAATTGATAGATTTCGGGAGAAACGCATCCAGC 60 15 AACGGCTCTCTCACGGCCCCTCCTTGCACCGAAGGTGTTGCGTGGTTTGTTATCGAAGAG 60 16 TTTGATTTCCGCCAGCTGTTTCGCAGAGACCTCCAGTGGCTCTTCGATAACAAACCACGC 60 17 ACAGCTGGCGGAAATCAAAAAACGTATGAAGAACTCTCCGAACCAACGCCCGGTGCAGCC 60 18 TTAACGGGTCTTTGCAGAAGATTTGATGATAACGGTGTTATAATCCGGCTGCACCGGGCG 60


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

added (9/13/13)- https://docs.google.com/file/d/0B_Gl3lMyhDsoYVNRdHI4ZjZnc2c/edit
 * Primer design results for pNIC-Bsa4 cloning (list sequences 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)

Forward Primer: 5’ TACTTCCAATCCATGAAAAAAACCTTCCTG 3’ 30 bp GC Content=36.7% 0mM Mg2+ Tm=58.1°C 1.5mM Mg2+ Tm=65.8ºC 2mM Mg2+ Tm=66.4ºC 4mM Mg2+ Tm=67.5ºC 6mM Mg2+ Tm=68.1ºC
 * Primer design results for 'tail' primers (this is just 2 sequences): -** added (9/13/13)

Reverse Primer: 5’ TATCCACCTTTACTGTTAACGGGTCTTTGCAGA 3’ 33 bp GC Content=42.4% 0mM Mg2+ Tm=62.4°C 1.5mM Mg2+ Tm=69.8ºC 2mM Mg2+ Tm=70.3ºC 4mM Mg2+ Tm=71.3ºC 6mM Mg2+ Tm=71.8ºC (9/14/13) Forward Primer: TACTTCCAATCCATGAAAAAAACTTTCCTGATCGCGC 37 bp GC Content: __%__ __ 2mM Mg2+ Tm: 71.5 Celsius __

__ Reverse Compliment (Downstream): __ __ TATCCACCTTTACTGACGGGTTTTCGCAGAAGA __ __ 33 bp __ __ GC Content: __% 2 mM Mg2+ Tm: 71.5 Celsius

http://www.medicinenet.com/helicobacter_pylori/page2.htm#what_does_h_pylori_cause_in_humans http://www.webmd.com/digestive-disorders/h-pylori-helicobacter-pylori
 * References:**

10/25/13 QasimA.

Enter the Template PDB ID: 1KOPB Enter the Save the Identities 37 % and Positives 50 % Enter the Molprobity scores for both the Template and the Model: Template: Clash score = 6.26 Molprobity score = 1.64 Model: Clash score= 16.3 Molprobity score = 2.90

10/31/13 Kelly K. Enter the Template PDB ID: 4G7A Enter the Save the Identities 39 % and Positives 55 % Ener the Molprobity scores for both the Template and the Model: Template: Clash score = 6.26 Molprobity score = 1.50 Model: Clash score = 29.99 Molprobity score = 3.23