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Keywords = human rhinovirus 3C protease cleavage

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12 pages, 1557 KiB  
Communication
Design of a Human Rhinovirus-14 3C Protease-Inducible Caspase-3
by Hanna J. Wagner and Wilfried Weber
Molecules 2019, 24(10), 1945; https://doi.org/10.3390/molecules24101945 - 21 May 2019
Cited by 5 | Viewed by 4920
Abstract
The engineering of enzymes for the purpose of controlling their activity represents a valuable approach to address challenges in both fundamental and applied research. Here, we describe and compare different design strategies for the generation of a human rhinovirus-14 (HRV14) 3C protease-inducible caspase-3 [...] Read more.
The engineering of enzymes for the purpose of controlling their activity represents a valuable approach to address challenges in both fundamental and applied research. Here, we describe and compare different design strategies for the generation of a human rhinovirus-14 (HRV14) 3C protease-inducible caspase-3 (CASP3). We exemplify the application potential of the resulting protease by controlling the activity of a synthetic enzyme cascade, which represents an important motif for the design of artificial signal transduction networks. In addition, we use our engineered CASP3 to characterize the effect of aspartate mutations on enzymatic activity. Besides the identification of mutations that render the enzyme inactive, we find the CASP3-D192E mutant (aspartate-to-glutamate exchange at position 192) to be inaccessible for 3C protease-mediated cleavage. This indicates a structural change of CASP3 that goes beyond a slight misalignment of the catalytic triad. This study could inspire the design of additional engineered proteases that could be used to unravel fundamental research questions or to expand the collection of biological parts for the design of synthetic signaling pathways. Full article
(This article belongs to the Special Issue Molecules for Biotechnologies)
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9 pages, 248 KiB  
Article
Over-expression, Rapid Preparation and Some Properties of C-terminal BARc Region in PICK1
by Hong Xiao, Yawei Shi, Jingming Yuan, Yuming Huang and Junhua Wang
Int. J. Mol. Sci. 2009, 10(1), 28-36; https://doi.org/10.3390/ijms10010028 - 27 Dec 2008
Cited by 3 | Viewed by 13195
Abstract
A DNA fragment encoding C-terminal BARc region (amino acids 128-416) of rat PICK1 (NP_445912 ) was inserted into a modified vector pMAL-s involving human rhinovirus 3C protease cleavage site to produce a recombinant plasmid, pMAL-s-barc. The construct can express the fusion [...] Read more.
A DNA fragment encoding C-terminal BARc region (amino acids 128-416) of rat PICK1 (NP_445912 ) was inserted into a modified vector pMAL-s involving human rhinovirus 3C protease cleavage site to produce a recombinant plasmid, pMAL-s-barc. The construct can express the fusion protein, MBP-BARc in the soluble form in E.coli. To remove the MBP tag, MBP-BARc purified from amylose beads was digested with human rhinovirus 3C protease and the cleavage efficiency is about 95% when the ratio of protein / enzyme (w/w) reaches 50:1, as analyzed on SDS-PAGE. The enzymatic reaction mixture was rapidly separated into two parts, MBP in the supernatant and BARc in the precipitate at the concentration of 1 M ammonium sulfate. In such case, the target protein BARc could be economically produced in a soluble state to be as the sample for measuring its biochemical function, for example, protein-protein interaction and protein-lipid combination. Full article
(This article belongs to the Section Biochemistry)
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