High-voltage isoelectric focusing with pharmalyte: Field strength and temperature distribution, zone sharpening, isoelectric spectra, and pI determinations

Låås, Torgny; Olsson, Ingmar; Söderberg, Linda

doi:10.1016/0003-2697(80)90212-2

Cited by 44 publications

(7 citation statements)

References 13 publications

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“…3). The overall temperature profile agreed with previously reported ones but the deviation was relatively small in our experiments, reflecting the lower field strength (1000 V/10 cm) used in our experiment as compared with previous data at 2300 V [14]. The actual distribution of the four acidic peptides which focused in this range of gel showed an effective temperature deviation of, at most, ±2.…”

Section: Determination Of Pi Of the Peptidessupporting

confidence: 90%

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Shimura

Wang

Matsumoto³

et al. 2000

Electrophoresis

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Section: Determination Of Pi Of the Peptidessupporting

confidence: 90%

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Shimura

Wang

Matsumoto³

et al. 2000

Electrophoresis

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“…The pHi value of BaFFase was determined by isoelectric focusing in thin slabs of both polyacrylamide and agarose. The method used to determine marker pI values was based on the procedure suggested by Laas et al 58…”

Section: Baffase Isoelectric Pointmentioning

confidence: 99%

“…Performing the assays in thin slabs of both polyacrylamide and agarose and following the procedure suggested by Laas et al 58 Source: By the author. Having the integrated elution curves, we prepared a calibration curve by plotting the logarithms of the known molecular weights of the six standard protein versus their respective Ve/Vo.…”

Section: Baffase Isoelectric Pointmentioning

confidence: 99%

Structural and enzymatic features of a recombinant β-fructofuranosidase from <i>Bifidobacterium adolescentis</i>

Mera¹

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MONSALVE ALAIN, M. Structural and enzymatic features of a recombinant βfructofuranosidase from Bifidobacterium adolescentis. 2016. 92 p. Dissertation (Master in Science) -Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, 2016.Despite the fact that Glycosyl Hydrolase Family 32 present 4 467 enzyme entries, only 14 of them have been characterized structurally. From the ten protein crystal structures deposited for Bifidobacterium adolescentis ATCC 15703 at PDB just one enzyme is related to the processing of non-digestible sugars and there is no structure of a β-fructofuranosidase. In this research we studied the biochemical properties and the structural features of a recombinant βfructofuranosidase (BaFFse) from the healthy gut bacteria B. adolescentis ATCC 15703 (gen BAD_1325) heterologously expressed in Escherichia coli Rosetta. The enzyme was purified by nickel ion affinity chromatography and molecular exclusion chromatography; the purification process was judged by denaturing SDS-PAGE gel. Sucrose was used as a substrate for the enzyme activity assays and the amount of reducing sugars, detected by Dinitrosalycilic acid, was taken as indicator of the optimum conditions of hydrolysis for the enzyme. BaFFase crystal, grown in PEG 8K 25% (w/v) and buffer MES 0.1M pH 6.5, was diffracted at 2.44 Å and processed using the CCP4 program package. The enzyme presented a classical four-stranded five-bladed β-propeller and a C-terminal β-sandwich characteristic from the GH 32 family; however, connected to the β-propeller through a loop of 38 residues, BaFFase also presented an N-terminal β-sandwich domain, which sequence (residues 3-100 from BaFFase) did not match with any protein sequence when aligned against PDB database.Assays with Gel filtration calibration, DLS and SAXS showed that the enzyme was a stable homodimer in solution. Based on the superposition of structures using the a βfructofuranosidase from B. longum KN29.1 we could deduced the three key aminoacids involved in the transferring of fructosyl moieties by BaFFase. A nucleophile attack is performed by the carboxylate of Asp 131, forming the fructose -BaFFase intermediate; Glu 375 donates a proton, acting as an acid base catalyst and Asp 269 stabilizes the transitions state in the fructosyl transferring activity. This is the first GH32 oligomeric enzyme belonging to the bacteria kingdom. We have described a novel additional β-sandwich domain for a GH32 enzyme that increases the region of contact to form a dimer. This is the first βfructofuranosidase crystal structure from the microorganism B. adolescentis ATCC 15703.

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“…PVP was widely used for cell lysis in many plants and macroalgae (Gygi et al 2000;Nagai et al 2008). Pharmalyte was also used in 2-DE based protein analysis to enhance protein solubility by preventing protein interactions that generate aggregation (Låås et al 1980). To test the effect of detergents, we added 0-5% Triton X-100, PVP, or pharmalyte.…”

Section: Effect Of Ph On Protein Expressionmentioning

confidence: 99%

An improved method of protein isolation and proteome analysis with Saccharina japonica (Laminariales) incubated under different pH conditions

Kim

et al. 2010

J Appl Phycol

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The brown alga Saccharina japonica is abundant on rocky coasts of Far East Asia, including Korea, Japan, and China. S. japonica produces high levels of compounds used in the food, cosmetic, and pharmaceutical industries. Thus, many studies have focused on the biosynthesis, extraction, purification, and application of carbohydrates, as well as biochemical features that yield cellular proteins. However, total protein isolation has proved difficult, due to viscous polysaccharides on the surface of S. japonica. To extract total proteins cleanly from S. japonica, we examined various lysis buffers and detergents for effective cell lysis and removal of polysaccharide. Lysis solution D (7 M urea, 4% [3-(3-cholami-dopropyl dimethylammonio) propanesulfonate], 2 M thio-urea, 100 mM dithiothreitol, 4% pharmalyte, 4% polyvinylpyrrolidone) achieved a comparatively high yield of protein extraction, with 12 mg of proteins purified per 1 g of dry weight of S. japonica. Proteins isolated using lysis solution D and subjected to two-dimension polyacrylamide gel electrophoresis generated more than 200 protein spots. Of these, 60 spots were analyzed by matrixassisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS. A database search revealed that these proteins include glyceraldehyde-3-phosphate dehydrogenase, tryptophan synthase α chain, 6-phosphogluconate dehydrogenase (6PGD), actin, phosphoglycerate kinase, elongation factor Tu, kinesin, fucoxanthin-chlorophyll a-c binding protein F precursor and ATP synthase subunit β. Many protein spots were unidentified. When S. japonica was incubated at different pH, tryptophan synthase α chain and variant surface glycoprotein 7 precursor were highly expressed at pH 7.5 and 9.5, respectively, whereas 6PGD and kinesin showed low expression at pH 9.5.

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High-voltage isoelectric focusing with pharmalyte: Field strength and temperature distribution, zone sharpening, isoelectric spectra, and pI determinations

Cited by 44 publications

References 13 publications

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Structural and enzymatic features of a recombinant β-fructofuranosidase from <i>Bifidobacterium adolescentis</i>

An improved method of protein isolation and proteome analysis with Saccharina japonica (Laminariales) incubated under different pH conditions

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High-voltage isoelectric focusing with pharmalyte: Field strength and temperature distribution, zone sharpening, isoelectric spectra, and pI determinations

Cited by 44 publications

References 13 publications

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Structural and enzymatic features of a recombinant &beta;-fructofuranosidase from <i>Bifidobacterium adolescentis</i>

An improved method of protein isolation and proteome analysis with Saccharina japonica (Laminariales) incubated under different pH conditions

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Structural and enzymatic features of a recombinant β-fructofuranosidase from <i>Bifidobacterium adolescentis</i>