Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Gardner, Qurratulann Afza; Younas, Hooria; Akhtar, Muhammad

doi:10.1016/j.bbapap.2012.09.004

Cited by 15 publications

(14 citation statements)

References 28 publications

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“…To confirm that the new domains do not influence binding between BPTI and trypsin, we determined the binding affinities by isothermal titration calorimetry (ITC). We observed that both the SUMO and K36 domains reduce the binding affinity, although the affinities with dissociation constants of circa 10 −8 m are still higher than trypsin with most of its substrates (Figure S24–S26) [51] …”

Section: Methodsmentioning

confidence: 89%

“…We observed that both the SUMO and K36 domains reduce the binding affinity, although the affinities with dissociation constants of circa 10 À8 m are still higher than trypsin with most of its substrates ( Figure S24-S26). [51] We determined the activity of trypsin from the turnover of its substrate N a -benzoyl-l-arginine ethyl ester (BAEE), which results in absorbance at 253 nm. This leads to complete digestion within 2 min, with a specific activity of 40 BAEE units mL À1 trypsin, while the presence of any of our BPTI derivatives completely inhibits the reaction.…”

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confidence: 99%

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Controlling Optical and Catalytic Activity of Genetically Engineered Proteins by Ultrasound

et al. 2020

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Ultrasound (US) produces cavitation-induced mechanical forces stretching and breaking polymer chains in solution. This type of polymer mechanochemistry is widely used for synthetic polymers, but not biomacromolecules, even though US is biocompatible and commonly used for medical therapy as well as in vivo imaging. The ability to control protein activity by US would thus be a major stepping-stone for these disciplines. Here, we provide the first examples of selective protein activation and deactivation by means of US. Using GFP as a model system, we engineer US sensitivity into proteins by design. The incorporation of long and highly charged domains enables the efficient transfer of force to the protein structure. We then use this principle to activate the catalytic activity of trypsin by inducing the release of its inhibitor. We expect that this concept to switch "on" and "off" protein activity by US will serve as a blueprint to remotely control other bioactive molecules.

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Section: Methodsmentioning

confidence: 89%

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confidence: 99%

Controlling Optical and Catalytic Activity of Genetically Engineered Proteins by Ultrasound

et al. 2020

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“…One microliter of sample was spotted on MALDI plate (MTP Anchor chip var. 384), dried, and analyzed in linear positive mode as detailed in Gardner et al (2013).…”

Section: Methodsmentioning

confidence: 99%

Effect of Milking Method, Diet, and Temperature on Venom Production in Scorpions

Tobassum

Tahir

Zahid

et al. 2018

Journal of Insect Science

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In the present study, two common buthid scorpions, i.e., Androctonus finitimus (Pocock, 1897) (Scorpiones: Buthidae) and Hottentota tamulus (Fabricus, 1798) (Scorpiones: Buthidae), were maintained in the laboratory for venom recovery. The aim of study was to compare the quantity and quality of venom extracted from scorpions by manual and electrical method. We also recorded the effect of diet and temperature on venom production. Results of our study revealed that electrical method yielded good quality and higher quantity of venom as compared to manual method. The quantity of venom by two studied species differed statistically. We recorded the effect of food on venom production by providing different prey items to the scorpions and found that grasshopper nymphs and adults were the best diet for the scorpions to get maximum yield of venom as compared to other prey types (house crickets, house flies, and moths). Production of venom and activity of scorpions was found to be associated with temperature. During winter season, venom recovery was comparatively low as compared to the hottest part of year; when venom milking and activity of scorpions both were increased.

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“…25 ml of the solubilized protein above (5 mg of protein /ml) was transferred to the refolding sink to 60 percent of the starting inclusion bodies. For the identification of properly refolded proteins, tryptic digestion of various interferon constructs was performed as described in [1] and MALDI mass spectrometric analysis of fragment containing correct disulphide bridges was performed in linear positive mode as described previously [19].…”

Section: Expression and Purification Of Interferon Derivativesmentioning

confidence: 99%

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

Ahsan

Gardner

Rashid

et al. 2018

Bioorganic Chemistry

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We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b at the N-terminal of interferon (b-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b at the C-terminal of interferon (Met-IFN-b-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b-chimera), resolved this problem and gave enhanced biological activity.

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Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Cited by 15 publications

References 28 publications

Controlling Optical and Catalytic Activity of Genetically Engineered Proteins by Ultrasound

Controlling Optical and Catalytic Activity of Genetically Engineered Proteins by Ultrasound

Effect of Milking Method, Diet, and Temperature on Venom Production in Scorpions

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

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