Purification and characterization of natural Ara h 8, the Bet v 1 homologous allergen from peanut, provides a novel isoform

2008 ◽  
Vol 389 (4) ◽  
pp. 415-423 ◽  
Author(s):  
Susanne Riecken ◽  
Buko Lindner ◽  
Arnd Petersen ◽  
Uta Jappe ◽  
Wolf-Meinhard Becker
Keyword(s):  
Birch Pollen ◽  
Diagnostic Procedures ◽  
Cross Reactivity ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Reactive Protein ◽  
Bet V 1 ◽  
Peanut Extract ◽  
Natural Counterpart

Abstract The peanut allergen Ara h 8 is an important allergen for birch pollen allergic patients because of the cross-reactivity to the homologous Bet v 1. As the existence of Ara h 8 has been shown at the cDNA level so far (AY328088) and the allergen has indirectly been detected as natural protein, it was the aim of our study to identify natural Ara h 8 in peanut extract and to develop a purification strategy. This was achieved using a unique combination of purification steps, including optimized extraction conditions, size exclusion and ion exchange chromatography and treatment of the interfering contaminants with iodo-acetic acid. A characterization of the protein by microsequencing showed discrepancies to the deduced amino acid sequence of AY328088. For this reason, we cloned and expressed a new Ara h 8 isoform from cDNA (EU046325). This IgE-reactive protein corresponds to the results of microsequencing, ESI-FTICR-MS and trypsin fingerprinting analysis of the authentic and purified nAra h 8. Apart from the ultimate use of recombinant allergens for diagnostic procedures, there is also a scientific need for the natural counterpart, as it represents an excellent reference point by which to compare protein characteristics and to standardize diagnostic and therapeutic allergens.

scholarly journals Purification and Biochemical Characterization of Alkaline Serine Protease from Caesalpinia bonducella

2010 ◽  
Vol 5 (6) ◽  
pp. 1934578X1000500
Author(s):  
Hidayatullah Khan ◽  
Irshad Ali ◽  
Arif-ullah Khan ◽  
Mushtaq Ahmed ◽  
Zamarud Shah ◽  
...  
Keyword(s):  
Serine Protease ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Alkaline Serine Protease ◽  
Protease Enzyme ◽  
Caesalpinia Bonducella

A high molecular weight serine protease has been purified to electrophoretic homogeneity from the seeds of Caesalpinia bonducella Flem. (Caesalpiniaceae) by the combination of size exclusion and ion exchange chromatography. About 524 fold purification was achieved with an overall recovery of 6.8%. The specific activity was found to be 86 U/mg/min at pH 8.0. The calculated Km and Vmax were 1.66 mg/mL and 496.68 units/min per mg of protein, respectively. The molecular mass was estimated to be about 63 kDa by sodium dodecyl sulfate PAGE. The enzyme showed optimum activity at pH 8.0 and exhibited its highest activity at 40°C. The enzyme was strongly inhibited by 2mM phenylmethylsulfonyl fluoride (PMSF), suggesting the presence of a serine residue at the active site. PMSF showed a pure competitive type of inhibition with the serine protease enzyme. It was observed that enzyme activity was enhanced in the presence of dications and was active against a variety of modified substrates and natural proteins.

scholarly journals Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

2009 ◽  
Vol 9 (1) ◽  
pp. 24 ◽  
Author(s):  
Martijn F Schenk ◽  
Jan HG Cordewener ◽  
Antoine HP America ◽  
Wendy PC van't Westende ◽  
Marinus JM Smulders ◽  
...  
Keyword(s):  
Birch Pollen ◽  
Bet V 1

Characterization of the Mechanism of Interaction between C-Reactive Protein and GPIIb/IIIa.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1517-1517
Author(s):  
Marian P. Brennan ◽  
Roisin D. Moriarty ◽  
Stephanie Arasu ◽  
Timothy J. Foster ◽  
Dermot Cox
Keyword(s):  
Platelet Adhesion ◽  
Size Exclusion ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Exclusion Chromatography ◽  
Mechanism Of Interaction ◽  
Platelet Spreading ◽  
Circulating Levels ◽  
Lamellipodia Formation

Abstract C-reactive protein (CRP) is a well established marker for inflammation, and a good predictor of coronary heart disease. It is also known to interact with the platelet FcγRIIa and to enhance the inhibition of platelet aggregation by aspirin by an unknown mechanism. CRP has also recently been demonstrated to compete for PAC-1 binding in collagen stimulated platelets, suggesting that CRP interacts with GPIIb/IIIa. In order to study the mechanism of interaction with platelets directly, we carried out platelet adhesion assays. We coated plates with recombinant CRP which was in the native pentameric form as confirmed by size exclusion chromatography. Platelets adhered to immobilized CRP and to immobilized fibrinogen to a similar extent. Adhesion to CRP and fibrinogen was inhibited by GPIIb/IIIa antagonists but not by antibody to the FcγRIIa (IV.3). Platelet adhesion to CRP was increased 5-fold when platelets were treated with 1 mM MnCl2. Adhesion of MnCl2 treated platelets was also inhibited by GPIIb/IIIa antagonists. When viewed by confocal microscopy, the adherent platelets displayed pseudopodia, but did not spread fully. Treatment of platelets with MnCl2 stimulated lamellipodia formation and full platelet spreading on CRP. Analysis of the exposed residues on the surface of the CRP crystal structure identified two RGD-like sequences, RQD and DGK. The peptides KRQDN and VDGKP derived from CRP inhibited platelet adhesion to fibrinogen suggesting that these sequences could be responsible for the interaction with GPIIb/IIIa. Our data demonstrates that CRP in the native pentameric form interacts with GPIIb/IIIa and that it has an increased affinity for the activated conformation of GPIIb/IIIa. This interaction is likely to be due to an interaction with the surface exposed KRQDN and VDGKP CRP sequences. We have previously demonstrated that CRP acts as a GPIIb/IIIa antagonist when in solution. Furthermore, pentameric CRP cannot support platelet adhesion under shear conditions. Thus it is likely that elevated circulating levels of soluble CRP reduces the overall thrombotic potential.

Molecular characterization of Dau c 1, the Bet v 1 homologous protein from carrot and its cross-reactivity with Bet v 1 and Api g 1

1999 ◽  
Vol 29 (6) ◽  
pp. 840-847 ◽  
Author(s):  
HOFFMANN-SOMMERGRUBER ◽  
O'RIORDAIN ◽  
AHORN ◽  
EBNER ◽  
DA CAMARA MACHADO ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Cross Reactivity ◽  
Homologous Protein ◽  
Bet V 1

scholarly journals Structural Characterization of Glycerophosphorylated and Succinylated Cyclic β-(1→2)-d-Glucan Produced by Sinorhizobium mliloti 1021

Polymers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2073
Author(s):  
Hyojeong Lee ◽  
Seonmok Kim ◽  
Yohan Kim ◽  
Seunho Jung
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Attenuated Total Reflection ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Chemical Structures ◽  
Maldi Tof ◽  
Surface Polysaccharides ◽  
Target Molecules

Rhizobia produces different types of surface polysaccharides. Among them, cyclic β-(1→2)-d-glucan is located in the periplasmic space of rhizobia and plays an important role in the adaptation of bacteria to osmotic adaptation. Cyclic β-(1→2)-d-glucan (CG), synthesized from Sinorhiozbbium meliloti 1021, has a neutral and anionic form. In the present study, we characterized the exact chemical structures of anionic CG after purification using size exclusion s (Bio-Gel P-6 and P-2) chromatography, and DEAE-Sephadex anion exchange chromatography. The exact structure of each isolated anionic CG was characterized using various analytical methods such as nuclear magnetic resonance (NMR), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and matrix associated laser desorption ionization-time of Flight (MALDI-TOF) mass spectrometry. The precise chemical structures of novel anionic CG molecules were elucidated by various NMR spectroscopic analyses, including 1H, 13C, 31P, and 2D HSQC NMR spectroscopy. As a result, we discovered that anionic CG molecules have either glycerophosphoryl or succinyl residues at C6 positions of a neutral CG. In addition, the results of MALDI-TOF mass spectrometric analysis confirmed that there are two types of patterns for anionic CG peaks, where one type of peak was the succinylated CG (SCG) and the other was glycerophospholated CG (GCG). In addition, it was revealed that each anionic CG has one to four substituents of the succinyl group of SCG and glycerophosphoryl group of GCG, respectively. Anionic CG could have potential as a cyclic polysaccharide for drug delivery systems and a chiral separator based on the complexation with basic target molecules.

Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery

2007 ◽  
Vol 51 (12) ◽  
pp. 1527-1536 ◽  
Author(s):  
Mirko A. Bollen ◽  
Aranzazu Garcia ◽  
Jan H. G. Cordewener ◽  
Harry J. Wichers ◽  
Johannes P. F. G. Helsper ◽  
...  
Keyword(s):  
Birch Pollen ◽  
Purification And Characterization ◽  
Bet V 1

Bet v 1142-156 is the dominant T-cell epitope of the major birch pollen allergen and important for cross-reactivity with Bet v 1–related food allergens

2005 ◽  
Vol 116 (1) ◽  
pp. 213-219 ◽  
Author(s):  
Beatrice Jahn-Schmid ◽  
Astrid Radakovics ◽  
Dirk Lüttkopf ◽  
Stephan Scheurer ◽  
Stefan Vieths ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Epitope ◽  
Birch Pollen ◽  
Cross Reactivity ◽  
Pollen Allergen ◽  
T Cell Epitope ◽  
Food Allergens ◽  
Bet V 1 ◽  
Birch Pollen Allergen

scholarly journals Purification and Characterization of Transglutaminase from Streptomyces sp. TTA 02 SDS 14

2016 ◽  
Vol 11 (3) ◽  
Author(s):  
Lia Siti Nur'amaliyah ◽  
Dewi Seswita Zilda ◽  
Nisa Rachmania Mubarik
Keyword(s):  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Phenacyl Bromide ◽  
Optimum Temperature ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Exclusion Chromatography ◽  
Sulfonyl Fluoride

Streptomyces sp. TTA 02 SDS 14 is a transglutaminase producing bacteria which previously had been  screened along with more than one hundred isolates. This research aimed to purify and characterize transglutaminase from this strain. Transglutaminase was purified from crude enzyme by ultrafiltration, Q-Sepharose ion exchange chromatography and Sepacryl S200 size exclusion chromatography sequentially, obtaining yield and purification fold of  1.36%  and 27 folds, respectively. The molecular weight of the purified transglutaminase was 72 kDa detected by zymogram gel electrophoresis. The optimum temperature and pH were 50°C and 6. The transglutaminase was stable at 45°C and could be activated in the presence of 5 mM and 10 mM of Na+, K+, Li+,Ca2+, Mg2+, BPB (4-bromo-phenacyl bromide), and IAA (iodo acetamide acid), but the activity was inhibited by  the presence of Cu+, Zn2+, and PMSF (phenyl methyl sulfonyl fluoride).

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