Isolation of soybean trypsin inhibitors by affinity chromatography on anhydrotrypsin-Sepharose 4B

Pusztai, Á.; Grant, George; Stewart, James; Watt, William B.

doi:10.1016/0003-2697(88)90418-6

Cited by 25 publications

(21 citation statements)

References 15 publications

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“…The behaviour of the nonhomologous soybean Kunitz and Bowman-Birk inhibitors on anhydrotrypsin affinity chromatography has been shown to reflect their association constants with enzymes [29]. The correlation between C-terminal processing of pea TI and increased affinity for target enzymes is important in the context of their antinutritional role as well as for the exploitation of TI for the protection of crops.…”

Section: Discussionmentioning

confidence: 99%

Multiple isoforms of Pisum trypsin inhibitors result from modification of two primary gene products

et al. 1995

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Characterization of Pisum (pea) seed trypsin inhibitors (TI) and their corresponding cDNAs indicates that the pea TI gene family contains two genes. The existence of multiple TI isoforms can be attributed to post-translational modifications of primary gene products. Post-translational processing at the Cterminus during the desiccation stage of seed development results in the appearance of TI isoforms with increased affinity for the target enzyme, trypsin.

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Section: Discussionmentioning

confidence: 99%

Multiple isoforms of Pisum trypsin inhibitors result from modification of two primary gene products

et al. 1995

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“…In order to purify a trypsin inhibitor we used an affinity chromatography with trypsin attached to cyanogen bromideactivated -Sepharose 4B column as described by Pusztai et al [16] generating one retained peak ( Figure 1A). This peak was applied onto a reversed-phase HPLC (Vydac C-18TP) and three peaks were eluted with a liner acetonitrile gradient ( Figure 1B).…”

Section: Trypsin Inhibitor Purification and Molecular Mass Analysesmentioning

confidence: 99%

Screening and Purification of a Novel Trypsin Inhibitor from Prosopis juliflora Seeds with Activity Toward Pest Digestive Enzymes

Sivakumar¹,

Franco²,

Tagliari³

et al. 2005

PPL

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Several pests are capable of decreasing crop production causing severe economical and social losses. Aiming to find novel molecules that could impede the digestion process of different pests, a screening of alpha-amylase and trypsin-like proteinase inhibitors was carried out in Prosopis juliflora, showing the presence of both in dry seeds. Furthermore, a novel trypsin inhibitor, with molecular mass of 13,292 Da, was purified showing remarkable in vitro activity against T. castaneum and C. maculatus.

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“…For binding a specific peptide we tested various binding and elution mobile phases and compared our results with those obtained with conditions already published. Acetate buffer (50 mM) at pH 5.0 containing 20 mM CaCl 2 [17,19,20,22] and 25 mM Tris buffer at pH 7.5 containing 0.125 M NaCl [25], respectively were used for affinity binding. From our results, it emerged that the type of buffer, ionic additives, and pH level are significant parameters for binding efficiency.…”

Section: Resultsmentioning

confidence: 99%

“…Acetate buffer (0.1 M, pH 5.0) with 20 mM CaCl 2 was found to be optimal. The elution of specific peptides could be achieved by decreasing the pH with 5 mM hydrochloric acid [16,19,22], 0.1 M formic acid [20], or 50 mM glycine buffer at pH 2.2 [25]. In terms of compatibility with MALDI-TOF-MS analysis, 0.1 M formic acid was successfully applied.…”

Section: Resultsmentioning

confidence: 99%

“…AHT has no affinity for free amino acids and/or peptides with Arg, Lys, and AECys presented elsewhere in the peptide molecule than at the C-terminus [22]. According to the literature the affinity ligand AHT, immobilized either on sepharose (AT-Sepharose) or agarose, was successfully used to trap peptides that corresponded to the products generated by the action of various trypsin-like proteases [16,18], biologically active peptides [21,23], trypsin inhibitors [24,25], or for recombinant proteins with low biological activity where commonly used affinity chromatography is not convenient [19].…”

Section: Introductionmentioning

confidence: 99%

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Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom‐up shotgun proteomics strategy

Korecká

Jankovičová

Křenková

et al. 2008

J of Separation Science

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Original PaperBioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategyWe report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG) -Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.

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Isolation of soybean trypsin inhibitors by affinity chromatography on anhydrotrypsin-Sepharose 4B

Cited by 25 publications

References 15 publications

Multiple isoforms of Pisum trypsin inhibitors result from modification of two primary gene products

Multiple isoforms of Pisum trypsin inhibitors result from modification of two primary gene products

Screening and Purification of a Novel Trypsin Inhibitor from Prosopis juliflora Seeds with Activity Toward Pest Digestive Enzymes

Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom‐up shotgun proteomics strategy

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