Serine-Threonine-Protein+Kinase+B+(Mycobacterium+tuberculosis)


 * *Target: ** Serine/Threonine-Protein Kinase B (PKNB)
 * *NCBI Gene #: **887072

__ http://www.ncbi.nlm.nih.gov/gene/887072 __

__ http://www.mayoclinic.org/diseases-conditions/tuberculosis/basics/risk-factors/con-20021761 __ Mycobacterium tuberculosis is an organism that causes the well-known disease, tuberculosis (TB). Specifically, M. tuberculosis is pathogenic towards humans and usually attacks the lungs, however can infect any part of the body. It is an airborne pathogen that can spread from one person to another. TB is one of the leading causes of death due to an infectious disease. M. tuberculosis is able to avoid intercellular killing and enter macrophages, thus a reason for its prevalence and severity. There are two forms of TB: latent TB infection and TB disease, where the M. bacterium is inactive in the body and does not cause harm or the bacteria is active and the immune system cannot fight it off, respectively. The target focused on, serine threonine kinase protein B is essential for cell division and cell shape since it is a transmembrane protein. [] []
 * *Protein ID: ** 5.08
 * *Organism: ** Mycobacterium tuberculosis
 * Etiologic Risk Group: **
 * 1) Weakened immune system
 * 2) Traveling or living in certain conditions or areas
 * 3) Poverty and substance abuse
 * * Disease Information : **

[] [] [] Multimer biological unit. Query 9 DRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREAQNAAALNH 68 D Y + ++G G L + ++ A+K +R L + S R+EA A + H Sbjct 2 DDYMVLRMIGEGSFGRALLVQHESSNQMFAMKEIR--LPKSFSNTQNSRKEAVLLAKMKH 59
 * Link to TDR Targets page (if present): **
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) **
 * Essentiality of this protein: ** Essential for cell survival because it plays a critical role in regulating cell division and cell wall synthesis. Essentiality was tested in experiments to determine the purpose of serine/threonine PKNB in M. tuberculosis cells.
 * Complex protein: ** not complex
 * Similarity to human: **
 * ** Score ** || ** Expect ** || ** Method ** || ** Identities ** || ** Positives ** || ** Gaps ** ||
 * 117 bits(292) || 1e-27 || Compositional matrix adjust. || 82/256(32%) || 129/256(50%) || 14/256(5%) ||

Query 69 PAIVAVYDTGEAETPAGPLPYIVMEYVDGVTL-RDIVHTEGPMTPKRAI-EVIADACQAL 126 P IVA ++ EAE G L YIVMEY DG L + I +G + P+ I C + Sbjct 60 PNIVAFKESFEAE---GHL-YIVMEYCDGGDLMQKIKQQKGKLFPEDMILNWFTQMCLGV 115

Query 127 NFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPE 186 N H+ ++HRD+K NI ++ VK+ DFG AR ++ N + +GT Y+ PE Sbjct 116 NHIHKKRVLHRDIKSKNIFLTQNGKVKLGDFGSARLLS---NPMAFACTYVGTPYYVPPE 172

Query 187 QARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARHEGLSAD 246 + +SD++SLGC+LYE+ T + PF +S ++ + V + I P H S + Sbjct 173 IWENLPYNNKSDIWSLGCILYELCTLKHPFQANSWKNLILK-VCQGCISPLPSH--YSYE 229

Query 247 LDAVVLKALAKNPENR 262 L +V + +NP +R Sbjct 230 LQFLVKQMFKRNPSHR 245

[]

__ http://www.brenda-enzymes.org/enzyme.php?ecno=2.7.11.1 __
 * Druggable Target: **0.7
 * *EC#: **2.7.11.1
 * Link to BRENDA EC# page: **
 * Enzyme Assay information: ** In Vitro Kinase Assays

=
// In Vitro Kinase Assays // —//In vitro// phosphorylation of each recombinant kinase (0.5 μg) was carried out for 30 min at 37 °C in a reaction mixture (20 μl) containing buffer P (25 m M Tris-HCl, pH 7.0, 1 m M dithiothreitol, 5 m M MgCl 2, 1 m M EDTA) with 200 μCi/ml [γ- 33 P]ATP corresponding to 65 n M (3000 Ci/mmol). After incubation, the reaction was stopped by adding sample buffer and heating the mixture at 100 °C for 5 min. =====

[]
 __ http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/invitro_kinase.xhtml#Radioactiveinvitrokinaseassays-Productsandcatalognumbers __
 * --- List cost and quantity of substrate reagents, supplier, and catalog # **
 * ~ Compound ||~ Specific activity (Ci/mmol) ||~ Rad. conc. (mCi/mL) ||~ Molar concentration (μM) ||~ EasyTides version containing dye in buffer (Shipped ambient, store at 2-8°C) ||~ Frozen version (Shipped on dry ice, store at -20°C) ||
 * ATP,[gamma- 32 P] || 10 || 2 || 200 ||  || [|BLU002/NEG002] ||
 * ^  || 3000 || 5 || 1.7 || [|BLU502H/NEG502H] || [|BLU002H/NEG002H] ||
 * ^  || 3000 || 10 || 3.3 || [|BLU502A/NEG502A] || [|BLU002A/NEG002A] ||
 * ^  || 6000 || 10 || 1.7 || [|BLU502Z/NEG502Z] || [|BLU002Z/NEG002Z] ||
 * ^  || 6000 || 150 || 25 ||   || [|NEG035C] ||
 * ATP,[gamma- 33 P] || 3000 || 10 || 3.3 || [|NEG602H] || [|NEG302H] ||
 * ATP,[gamma- 33 P] for HTS (stabilized) || 3000 || 10 || 3.3 || [|NEG602K] ||  ||
 * GTP,[gamma- 32 P] || 6000 || 10 || 1.7 || [|BLU504Z/NEG504Z] || BLU004Z/NEG004Z ||
 * ATP,[gamma- 35 S] || 25-100 || 12.5 || 125-500 ||  || [|NEG027] ||
 * || 1250 || 12.5 || 10 ||  || [|NEG027H] ||

-- PDB # 1MRU __ http://www.rcsb.org/pdb/explore/explore.do?structureId=1MRU __ ** Current Inhibitors: ** ATP kinase inhibitors  __ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158675/ __ Method used in an experiment by Yosseff Av Gay:
 * Structure: **
 * Expression Information: ** Expressed in bacteria cells, such as Corynebacterium glutamicum.
 * Purification Method : **

=
Competent cells of //E. coli //BL21(DE3) were prepared according to the CaCl 2  method ( [|30] ) and were transformed by the heat shock method for 2 min at 42°C with 100 ng of pYA102. The transformed //E. coli // cells were then plated onto LB agar supplemented with ampicillin (100 μg/ml). Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml). After overnight incubation at 37°C with shaking, the individual cultures were diluted 1:100 in the same medium and incubation was continued at 37°C with shaking. Isopropyl-β- d -thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM when the optical density at 600 nm reached 0.6. Cultures were centrifuged at 5,000 × //g // for 15 min at room temperature, and pellets were lysed in B-Per (Pierce) bacterial protein extraction reagent. Proteins were separated by centrifugation (15,000 × //g //<span style="background-color: #ffffff; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">, 4°C, 15 min) into soluble and insoluble fractions. PknB inclusion bodies contained in the insoluble fractions were purified from //<span style="font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">E. coli //<span style="background-color: #ffffff; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">membrane proteins by washing in a solution of 10% B-Per reagent and centrifugation (45,000 × //<span style="font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">g //<span style="background-color: #ffffff; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">, 90 min, 4°C). PknB was separated by sodium dodecyl sulfate–7.5% polyacrylamide electrophoresis (SDS-PAGE) and stained with Coomassie blue or transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The N-terminal amino acid sequence was verified after electrophoresis of samples in SDS-PAGE gels and electroblotting onto PVDF membranes. Edman degradation was performed, and the sequence of the first 10 amino acids from the NH <span style="font-family: 'Times New Roman',stixgeneral,serif; font-size: 0.8461em; vertical-align: baseline;">2 <span style="background-color: #ffffff; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;"> terminus was determined at the University of British Columbia Protein Sequencing Laboratory. In order to obtain soluble protein, PknB inclusion bodies were resuspended in 1× phosphate-buffered saline (PBS) (pH 7.4) and slowly added drop-wise to a solution of 16 M urea and 2 M dithiothreitol (DTT) to make a final concentration of 8 M urea and 1 M DTT. Soluble PknB was then dialyzed via a Spectra/Por 8000 cellulose membrane (VWR Scientific) against 200 volumes of 1× Tris-buffered saline (pH 7.4) at 4°C for 16 to 24 h. The sample was then centrifuged for 15 min at 4°C and 15,000 × //<span style="font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">g //<span style="background-color: #ffffff; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;"> (Baxter), and approximately 20 mg of protein was loaded onto a 50-ml Macro-Prep SE agarose size-exclusion column (Bio-Rad), which was used as a desalting column. Proteins were eluted over time at 4°C with a size-exclusion buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM DTT, and 0.01 mM EDTA ( <span style="color: #642a8f; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">[|18] <span style="background-color: #ffffff; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">). The purity of PknB was tested by subjecting samples to SDS-PAGE followed by Coomassie blue staining. SDS-PAGE gels were prepared by the method of Laemmli ( <span style="color: #642a8f; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">[|17] <span style="background-color: #ffffff; font-family: 'Times New Roman',stixgeneral,serif; font-size: 16px;">). The gels were stained with Coomassie blue R-250 or silver stain. Protein concentrations were determined by the Bradford protein assay reagent =====

__ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC96941/ __

MTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREA QNAAALNHPAIVAVYDTGEAETPAGPLPYIVMEYVDGVTLRDIVHTEGPMTPKRAIEVIA DACQALNFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTA QYLSPEQARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARH EGLSADLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERTSLLSSA AGNLSGPRTDPLPRQDLDDTDRDRSIGSVGRWVAVVAVLAVLTVVVTIAINTFGGITRDV QVPDVRGQSSADAIATLQNRGFKIRTLQKPDSTIPPDHVIGTDPAANTSVSAGDEITVNV STGPEQREIPDVSTLTYAEAVKKLTAAGFGRFKQANSPSTPELVGKVIGTNPPANQTSAI TNVVIIIVGSGPATKDIPDVAGQTVDVAQKNLNVYGFTKFSQASVDSPRPAGEVTGTNPP AGTTVPVDSVIELQVSKGNQFVMPDLSGMFWVDAEPRLRALGWTGMLDKGADVDAGGSQH NRVVYQNPPAGTGVNRDGIITLRFGQ <span style="background-color: #ffffff; color: #403838; font-family: 'Lucida Sans Unicode',Arial,'Lucida Grande',Tahoma,Verdana,Helvetica,sans-serif;">11,700 M <span style="color: #403838; font-family: 'Lucida Sans Unicode',Arial,'Lucida Grande',Tahoma,Verdana,Helvetica,sans-serif; font-size: 0.85em;">−1 <span style="background-color: #ffffff; color: #403838; font-family: 'Lucida Sans Unicode',Arial,'Lucida Grande',Tahoma,Verdana,Helvetica,sans-serif;"> cm <span style="color: #403838; font-family: 'Lucida Sans Unicode',Arial,'Lucida Grande',Tahoma,Verdana,Helvetica,sans-serif; font-size: 0.85em;">−1 __ http://jb.asm.org/content/191/13/4056.full __
 * Image of protein (PyMol with features delineated and shown separately): **
 * *Amino Acid Sequence: **
 * *Length of your protein in Amino Acids: ** 626 amino acids
 * Molecular Weight of your protein in kiloDaltons: ** 66.51 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength: **
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html).



atgaccaccccttcccacctgtccgaccgctacgaacttggcgaaatccttggatttggg ggcatgtccgaggtccacctggcccgcgacctccggttgcaccgcgacgttgcggtcaag gtgctgcgcgctgatctagcccgcgatcccagtttttaccttcgcttccggcgtgaggcg caaaacgccgcggcattgaaccaccctgcaatcgtcgcggtctacgacaccggtgaagcc gaaacgcccgccgggccattgccctacatcgtcatggaatacgtcgacggcgttaccctg cgcgacattgtccacaccgaagggccgatgacgcccaaacgcgccatcgaggtcatcgcc gacgcctgccaagcgctgaacttcagtcatcagaacggaatcatccaccgtgacgtcaag ccggcgaacatcatgatcagcgcgaccaatgcagtaaaggtgatggatttcggcatcgcc cgcgccattgccgacagcggcaacagcgtgacccagaccgcagcagtgatcggcacggcg cagtacctgtcacccgaacaggcccggggtgattccgtcgacgcccgatccgatgtctat tccttgggctgtgttctttatgaagtcctcaccggggagccacctttcaccggcgactca cccgtctcggttgcctaccaacatgtgcgcgaagacccgatcccaccttcggcgcggcac gaaggcctctccgccgacctggacgccgtcgttctcaaggcgctggccaaaaatccggaa aaccgctatcagacagcggcggagatgcgcgccgacctggtccgcgtgcacaacggtgag ccgcccgaggcgcccaaagtgctcaccgatgccgagcggacctcgctgctgtcgtctgcg gccggcaaccttagcggtccgcgcaccgatccgctaccacgccaggacttagacgacacc gaccgtgaccgcagcatcggttcggtgggccgttgggttgcggtggtcgccgtgctcgct gtgctgaccgtcgtggtaaccatcgccatcaacacgttcggcggcatcacccgcgacgtt caagttcccgacgttcggggtcaatcctccgccgacgccatcgccacactgcaaaaccgg ggcttcaaaatccgcaccttgcagaagccggactcgacaatcccaccggaccacgttatc ggcaccgacccggccgccaacacgtcggtgagtgcaggcgacgagatcacagtcaacgtg tccaccggacccgagcaacgcgaaatacccgacgtctccacgctgacatacgccgaagcg gtcaagaaactgactgccgccggattcggccgcttcaagcaagcgaattcgccgtccacc ccggaactggtgggcaaggtcatcgggaccaacccgccagccaaccagacgtcggccatc accaatgtggtcatcatcatcgttggctctggtccggcgaccaaagacattcccgatgtc gcgggccagaccgtcgacgtggcgcagaagaacctcaacgtctacggcttcaccaaattc agtcaggcctcggtggacagcccccgtcccgccggcgaggtgaccggcaccaatccaccc gcaggcaccacagttccggtcgattcagtcatcgaactacaggtgtccaagggcaaccaa ttcgtcatgcccgacctatccggcatgttctgggtcgacgccgaaccacgattgcgcgcg ctgggctggaccgggatgctcgacaaaggggccgacgtcgacgccggtggctcccaacac aaccgggtcgtctatcaaaacccgccggcggggaccggcgtcaaccgggacggcatcatc acgctgaggttcggccagtag
 * *CDS Gene Sequence: **


 * *GC% Content for gene: ** 63.3%

__ http://www.genomicsplace.com/cgi-bin/gc_calculator.pl __

-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **
 * *GC% Content for gene (codon optimized): **
 * 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): **