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Pertussis purification

Veerada Raksanoh, Panchika Prangkio, Chompounoot Imtong, Niramon Thamwiriyasati, Kittipong Suvarnapunya, Lalida Shank, Chanan Angsuthanasombat
Previously, we demonstrated that the ∼130-kDa CyaA-hemolysin (CyaA-Hly, Met482 -Arg1706 ) from Bordetella pertussis was palmitoylated at Lys983 when co-expressed with CyaC-acyltransferase in Escherichia coli, and thus activated its hemolytic activity. Here, further investigation on a possible requirement of the N-terminal hydrophobic region (HP, Met482 -Leu750 ) for toxin acylation was performed. The ∼100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala751 -Arg1706 ) containing the Lys983 -acylation region (AR, Ala751 -Gln1000 ), but lacking HP, was co-produced with CyaC in E...
May 23, 2018: Biochemical and Biophysical Research Communications
Zenglan Li, Yan Zhang, Qi Wang, Zhengjun Li, Yongdong Liu, Songping Zhang, Guifeng Zhang, Guanghui Ma, Jian Luo, Zhiguo Su
Development of acellular pertussis vaccine (aPV) requires purification of several components from Bordetella pertussis. While the components pertussis toxin (PT) and filamentous hemagglutinin (FHA) have been successfully purified, the third component, pertactin, proves to be a difficult target due to its very low concentration. In order to solve its purification problem, we performed the surface potential analysis with GRASP2 program. The results demonstrated that there are two major charge patches, one negative and one positive, which are located separately on this linear protein...
July 25, 2016: Vaccine
Vanessa Zavatti, Hector Budman, Raymond Legge, Melih Tamer
Fluorescence spectroscopy in combination with multivariate statistical methods was employed as a tool for monitoring the manufacturing process of pertactin (PRN), one of the virulence factors of Bordetella pertussis utilized in whopping cough vaccines. Fluorophores such as amino acids and co-enzymes were detected throughout the process. The fluorescence data collected at different stages of the fermentation and purification process were treated employing principal component analysis (PCA). Through PCA, it was feasible to identify sources of variability in PRN production...
June 2016: Bioprocess and Biosystems Engineering
Josephina Hendrix, Timothy Read, Jean-Francois Lalonde, Phillip K Jensen, William Heymann, Elijah Lovelace, Sarah A Zimmermann, Michael Brasino, Joseph Rokicki, Robin D Dowell
We have developed a simple system for tagging and purifying proteins. Recent experiments have demonstrated that RTX (Repeat in Toxin) motifs from the adenylate cyclase toxin gene (CyaA) of B. pertussis undergo a conformational change upon binding calcium, resulting in precipitation of fused proteins and making this method a viable alternative for bioseparation. We have designed an iGEM Biobrick comprised of an RTX tag that can be easily fused to any protein of interest. In this paper, we detail the process of creating an RTX tagged version of the restriction enzyme EcoRI and describe a method for expression and purification of the functional enzyme...
December 19, 2014: ACS Synthetic Biology
Michaela B C Kilander, Jenny Dahlström, Gunnar Schulte
The WNT receptors of the Frizzled family comprise ten mammalian isoforms, bind WNT proteins and mediate downstream signaling to regulate stem cell fate, neuronal differentiation, cell survival and more. WNT-induced signaling pathways are either β-catenin-dependent or -independent, thereby dividing the 19 mammalian WNT proteins into two groups. So far hardly any quantitative, pharmacological information is available about WNT-FZD interaction profiles, affinities or mechanisms of signaling specification through distinct WNT/FZD pairings...
September 2014: Cellular Signalling
An-Guo Liu, Hua-Zhang Li, Xing-Ke Yan, Tian-You He, Li-Li Kan, Jun-Yan Wang, Li-Li Dong
OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Sanyin" acupoints (bilateral "Jiayin" and "Zhongyin") on the cellular immune function of rats with chronic abacterial prostatitis (CAP), so as to explore its mechanism underlying relieving CAP. METHODS: Forty male Wistar rats were randomly divided into four groups: control, model, medication (Cernilton) and EA (n = 10 rats/group). The CAP model was made by injection of allogeneic prostatein purification liquid + Freund's complete adjuvant (1:1) and diphtheria, pertussis, tetanus (DPT) vaccine (0...
June 2013: Zhen Ci Yan Jiu, Acupuncture Research
Emmanuel Kostakis, Conor Smith, Ming-Kuei Jang, Stella C Martin, Kyle G Richards, Shelley J Russek, Terrell T Gibbs, David H Farb
N-methyl D-aspartate (NMDA) receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies have revealed reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed-onset potentiation whereby PregS approximately doubles the cell's response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS...
August 2013: Molecular Pharmacology
Hossein Asgarian-Omran, Ali Akbar Amirzargar, Mohammad Arjmand, Mohammadreza Eshraghian, Behrooz Nikbin, Saeid Eshraghi, Marzieh Mahdavi, Jalal Khoshnoodi, Mahmood Jeddi-Tehrani, Hodjatallah Rabbani, Fazel Shokri
BACKGROUND: Filamentous hemagglutinin (FHA) is one of the most important immunoprotective antigens of Bordetella pertussis (B. pertussis) and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated. METHODS: Three overlapping coding sequences of FHA antigen were amplified from B. pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli (E...
January 2013: Avicenna Journal of Medical Biotechnology
Oren Shur, Kevin Dooley, Mark Blenner, Matthew Baltimore, Scott Banta
Typically, chromatography is the most costly and time-consuming step in protein purification. As a result, alternative methods have been sought for bioseparations, including the use of stimulus-responsive tags that can reversibly precipitate out of solution in response to the appropriate stimulus. While effective, stimulus-responsive tags tend to require temperature changes or relatively harsh buffer conditions to induce precipitation. Here we describe a synthetic peptide, based on the natural repeat-in-toxin (RTX) domain that undergoes gentler calcium-responsive, reversible precipitation...
April 2013: BioTechniques
Li-na Han, Tie-ling Li, Guo-lei Ding, Jian-wei Liu, Yu Ding, Ya-jing Zhang
OBJECTIVE: To construct the recombinant plasmid of human cardiac C protein (CCP) peptide with immunogenicity and to express, purification and renature fusion protein. The fusion protein was injected to Lewis rats to establish experimental autoimmune myocarditis (EAM) model. METHODS: Total RNA was extracted from human heart and used as the template for reverse transcriptase-directed cDNA synthesis. The cDNA was then amplified by polymerase chain reaction (PCR) using oligonucleotide primers specific for CCP peptide with immunogenicity...
August 2012: Zhonghua Xin Xue Guan Bing za Zhi
Oren Shur, Jun Wu, Donald M Cropek, Scott Banta
Intrinsically disordered peptides (IDPs) have recently garnered much interest because of their role in biological processes such as molecular recognition and their ability to undergo stimulus-responsive conformational changes. The block V repeat-in-toxin motif of the Bordetella pertussis adenylate cyclase is an example of an IDP that undergoes a transition from a disordered state to an ordered beta roll conformation in the presence of calcium ions. In solution, a C-terminal capping domain is necessary for this transition to occur...
May 2011: Protein Science: a Publication of the Protein Society
Anne E Boyer, Maribel Gallegos-Candela, Renato C Lins, Zsuzsanna Kuklenyik, Adrian Woolfitt, Hercules Moura, Suzanne Kalb, Conrad P Quinn, John R Barr
Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins...
2011: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Erich H Schneider, Roland Seifert
The Sf9 cell/baculovirus expression system is widely used for high-level protein expression, often with the purpose of purification. However, proteins may also be functionally expressed in the defined Sf9 cell environment. According to the literature, the pharmacology of G-protein-coupled receptors (GPCRs) functionally reconstituted in Sf9 cells is similar to the receptor properties in mammalian cells. Sf9 cells express both recombinant GPCRs and G-proteins at much higher levels than mammalian cells. Sf9 cells can be grown in suspension culture, providing an inexpensive way of obtaining large protein amounts...
December 2010: Pharmacology & Therapeutics
S Wurster, R Nakov, C Allgaier, G Hertting
The effect of the sulfhydryl alkylating agent N-ethylmaleimide on the modulation of potassium-evoked [(3)H]noradrenaline release via inhibitory presynaptic receptors was studied using synaptosomes from rabbit hippocampus. Dose-response curves for the ?(2)-adrenoceptor agonist clonidine, the preferential ?-opioid receptor agonist ethylketocyclazocine and the A(1)-adenosine receptor agonist (?)phenylisopropyladenosine were compared to the effects of these agonists after pretreatment of [(3)H]noradrenaline loaded synaptosomes with N-ethylmaleimide (2 ?M) for 15 min...
1990: Neurochemistry International
R Brodzik, S Spitsin, N Pogrebnyak, K Bandurska, C Portocarrero, K Andryszak, H Koprowski, M Golovkin
The current diphtheria-tetanus-pertussis (DTP) pediatric vaccine is produced from the corresponding pathogenic bacteria Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis; five injected doses of DTaP (acellular) vaccine are required for every child in the standard US vaccination schedule. Because the vaccine is derived from native live sources, adverse effects are possible and production is complex and costly. To address issues of safety, ease of renewability and expense, we used recombinant technology in an effort to develop a subunit DPT vaccine derived in non-pathogenic plant expression systems...
June 8, 2009: Vaccine
Ji-Fu Wei, Xiao-Long Wei, Ya-Zhen Mo, Shao-Heng He
BACKGROUND: It has been recognized that phospholipase A2 (PLA2) is a crucial component of snake venom, which contributes greatly to snake venom induced inflammation in man. However, the mechanisms through which N49 PLA2 provoke inflammation remain unclear. Recently, a N49 PLA2, TM-N49 from Protobothrops mucrosquamatus crude venom was characterized in our laboratory. Since the purification procedure developed is able to supply us with relatively large quantity of highly purified TM-N49, we investigated the ability of TM-N49 in induction of inflammation...
April 28, 2009: BMC Immunology
Hidehito Mukai, Tetsuo Seki, Hiroko Nakano, Yoshinori Hokari, Toshifumi Takao, Masanori Kawanami, Hiroyuki Tsukagoshi, Hirokazu Kimura, Yoshiaki Kiso, Yasutsugu Shimonishi, Yoshisuke Nishi, Eisuke Munekata
Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide. Structural analyses indicated that the primary structure of this peptide is formyl-Met-Thr-Asn-Ile-Arg-Lys-Ser-His-Pro-Leu-Met-Lys-Ile-Ile-Asn, which is identical to that of the N-terminal pentadecapeptide of porcine mitochondrial cytochrome b; we therefore named the newly isolated peptide "mitocryptide-2" (MCT-2), since we have recently purified and identified mitocryptide-1, a different class of a neutrophil-activating peptide...
April 15, 2009: Journal of Immunology: Official Journal of the American Association of Immunologists
Karl Brillet, Ahmed Meksem, Emmanuelle Lauber, Cornelia Reimmann, David Cobessi
Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms...
April 2009: Acta Crystallographica. Section D, Biological Crystallography
Tie Wu, Jingxiu Bi, Yongdong Huang, Yan Zhang, Lijing Sun, Chunbao Sun, Zhiguo Su
The low recovery of pertussis toxin (PT) and the low resolving efficiency of chromatography, due to the instability of PT in low salt condition, are the main challenges for its purification. We aplied 2 mol/L urea to prevent the aggregation and disassociation of PT during the purification by ion-exchange chromatography (IEC) and gel filtration chromatography (GFC). The effect of urea on the purification of PT was studied by ELISA assay and non-reduced SDS-PAGE. The activity recoveries of PT and filamentous hemagglutinin (FHA) in IEC and GFC, the resolution efficiency in GFC and the purities of PT and FHA were improved obviously by adding 2 mol/L urea in the mobile phase...
July 2008: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Julio O Ortiz, Mauricio Rodríguez-Lanetty, José Bubis
Polypeptides of approximately 39, 36 and <or= 14.4 kDa remained tightly bound to illuminated Hydrochoerus hydrochaeris retinal rod outer segment (ROS) membranes following extensive isotonic and hypotonic washes, and were specifically released in the presence of GTP. These results identified them as the alpha-, beta-and gamma-subunits of transducin (T). Once purified to homogeneity by anion-exchange chromatography, capybara T showed light-dependent beta,gamma-imido-guanosine 5'-triphosphate (GMPPNP) binding and GTPase activities in the presence of bovine rhodopsin, was recognized by anti-bovine T polyclonal antibodies, and was ADP-ribosylated by pertussis toxin...
January 2008: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology
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