Expression of chymotrypsin(ogen) in the thioredoxin reductase deficient mutant strain of Escherichia coli AD494(DE3) and purification via a fusion product with a hexahistidine-tail

Verheyden, Gert; Volckaert, Guido; Engelborghs, Yves

doi:10.1016/s0378-4347(99)00365-5

Cited by 5 publications

(4 citation statements)

References 42 publications

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“…In the case of each mutant it was checked and verified that they are sterically realizable. The choice for rat‐chymotrypsin (WT) instead of the bovine variant in the preparation of the mutants was due to the availability of the rat gene sequence in contrast to the bovine variant and because of cloning and purification reasons (Verheyden et al 2000). The role of the α‐propeptide is discussed elsewhere (G. Verheyden, J. Mátrai, P. Krüger, and Y. Engelborghs, unpubl.…”

Section: Resultsmentioning

confidence: 99%

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A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants

et al. 2004

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The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of ␣-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.

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Section: Resultsmentioning

confidence: 99%

“…The low pH prevents autodigestion, and the Ca 2+ ions prevent dimerization (Aune et al 1971; Stoesz and Lumry 1978; Shearwin and Winzor 1990). The purity of the preparates was analyzed by SDS polyacrylamide gel electrophoresis (Verheyden et al 2000).…”

Section: Methodsmentioning

confidence: 99%

A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants

et al. 2004

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“…The use of heterologous expression systems has demonstrated remarkable advantages over the traditional biochemical approach (Dimopoulos et al, 1996 lenga et al, 2001;Hanquier et al, 2003;Nakajima et al, 2003). Otherwise, despite the benefits of recombinant enzyme production, the trypsin expression in E. coli heterologous systems has also shown a common self-digestion enzyme and a multiple disulfide bound formation, due to the reducing environment in E. coli bacteria cytoplasm (Verheyden et al, 2000). Novel strategies have been applied to solving these difficulties, which included periplasmic expression, a known oxidant environment (Vasquez et al, 1989;Fukuoka et al, 2002), the use of a thioredoxin redutase deficient mutant strain (Verheyden et al, 2000;Prinz et al, 1997), and cytosolic expression followed by the use of oxidating buffers, as developed in this work (Shan et al, 2003;Hohenblum et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus

Magalhães

Fragoso

Souza

et al. 2007

Arch Insect Biochem Physiol

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The Mexican bean weevil, Zabrotes subfasciatus, feeds on several seeds such as Vigna unguiculata, Phaseolus vulgaris, and Pisum sativum, causing severe crop losses. This ability to obtain essential compounds from different diets could possibly be explained due to a wide variability of digestive proteinases present in the weevil's midgut. These may improve digestion of many different dietary proteins. Coleopteran serine-like proteinases have not been thoroughly characterized at the molecular level. In this report, a full-length cDNA encoding a trypsin-like protein, named ZsTRYP, was isolated from Z. subfasciatus larvae using RT-PCR, 5' and 3' RACE techniques. The quantitative real-time PCR analysis strongly correlated the Zstryp transcript accumulation to the major feeding developmental larval stage. Zstryp cDNA was subcloned into pET101 vector and expressed in a Escherichia coli BL21(DE3) strain. Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was used to purify a 29.0-kDa recombinant enzyme. The purified ZsTRYP was then assayed with several synthetic peptide substrates and also challenged with different inhibitors. The biochemical data allowed us to classify ZsTRYP as a trypsin. Moreover, homology modeling analysis indicated a typical trypsin structural core and a conserved catalytic triad (His(41), Asp(86), and Ser(182)).

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“…That is why the already cloned and very homologous in sequence (>80%) rat chymotrypsin(ogen) was used instead (Fig. 1; Verheyden et al 2000). In addition, the pro‐peptide (A‐chain) of chymotrypsin was substituted by the one of trypsinogen in order to provide a higher expression level and a more stable zymogen.…”

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confidence: 99%

Simulation of the activation of α‐chymotrypsin: Analysis of the pathway and role of the propeptide

et al. 2004

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Abstract␣-Chymotrypsin undergoes a reversible conformational change from an inactive chymotrypsinogen-like structure at high pH to an active conformation at neutral pH. In order to gain insight into this process on a structural level, we applied molecular dynamics and targeted molecular dynamics simulations in aqueous environment on the activation and inactivation processes of three different types of chymotrypsin. These are the wild-type bovine chymotrypsin containing the propeptide and the bovine and rat chymotrypsin lacking the propeptide. From these simulations, the importance of the propeptide and of the sequence differences between the rat and bovine variants from the viewpoint of activation could be evaluated and compared with previous fluorescence stopped flow results. The obtained results show the unambiguous influence of the propeptide on the explored conformational space, whereas the sequence differences between bovine and rat chymotrypsin play a minor role. The main features of activation are present in both the wild type and the variant lacking the propeptide, despite the fact that different parts of the conformational space were explored. The comparison of all trajectories shows that particular amino acid residues, such as 17, 18, 19, 187, 217, 218, and 223, undergo large dihedral transitions during the activation process, suggesting a role as hinge residues during the conformational change.Keywords: conformational change; molecular dynamics simulation; targeted molecular dynamics; pathway calculation; chymotrypsinogen; chymotrypsin; A-chain (propeptide); fluorescence stopped flow Supplemental material: see www.proteinscience.org Conformational changes play an essential role in the modulation of biological activity in proteins. Proteases are synthesized as inactive proenzymes, zymogens, and perform a series of conformational changes toward their active conformation in order to provide a site-and time-specific regulation of their proteolytic activity. The inactive protein precursors often consist of the intact protease with an N-terminal extension or prosegment, stabilizing the inactive enzyme conformation. Activation of the enzyme is then accomplished by limited cleavage of the propeptide in the zymogen. A typical example is the activation of chymotrypsinogen to chymotrypsin occurring after tryptic cleavage of the peptide bond between amino acid residues 15 and 16. This cleavage triggers a series of conformational changes that activates the enzyme (Bode et al. 1978;Bode 1979;Wang et al. 1985;Wroblowski et al. 1997). It allows the formation of a salt bridge between the newly released Ile 16 N terminus and the Asp 194 carboxyl group. This activation of the zymogen can also be mimicked by a pH jump from an inactive chymotrypsinogen-like structure at high pH to an active conformation around pH 7 and so can be followed spectroscopically (Fersht and Renard 1974 Abbreviations: rms, root mean square; MD, molecular dynamics; rmsd, root mean square deviation; TMD, targeted molecular dynamics; ⌬A-chymotrypsi...

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Expression of chymotrypsin(ogen) in the thioredoxin reductase deficient mutant strain of Escherichia coli AD494(DE3) and purification via a fusion product with a hexahistidine-tail

Cited by 5 publications

References 42 publications

A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants

A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants

Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus

Simulation of the activation of α‐chymotrypsin: Analysis of the pathway and role of the propeptide

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