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  4. A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein
 
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A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

Publikationstyp
Journal Article
Date Issued
2015-09-22
Sprache
English
Author(s)
Heyde, Amélie von der  
Lockhauserbäumer, Julia  
Uetrecht, Charlotte  
Elleuche, Skander  
Institut
Technische Mikrobiologie V-7  
TORE-URI
http://hdl.handle.net/11420/9812
Journal
Applied microbiology and biotechnology  
Volume
99
Issue
18
Start Page
7613
End Page
7624
Citation
Applied Microbiology and Biotechnology 99 (18): 7613-7624 (2015-09-22)
Publisher DOI
10.1007/s00253-015-6689-8
Scopus ID
2-s2.0-84939569153
PubMed ID
26026939
Extein amino acid residues around the splice site junctions affect the functionality of inteins. To identify an optimal sequence context for efficient protein splicing of an intein from the thermoacidophilic archaeon Picrophilus torridus, single extein amino acid residues at the splice site junctions were continuously deleted. The construction of a set of different truncated extein variants showed that this intein tolerates multiple amino acid variations near the excision sites and exhibits full activity when −1 and +1 extein amino acid residues are conserved in an artificial GST-intein-HIS fusion construct. Moreover, splicing of the recombinant intein took place at temperatures between 4 and 42 °C with high efficiency, when produced in Escherichia coli. Therefore, structural model predictions were used to identify optimal insertion sites for the intein to be embedded within a hemicellulase from the psychrophilic bacterium Pseudoalteromonas arctica. The P. torridus intein inserted before amino acid residue Thr75 of the reporter enzyme retained catalytic activity. Moreover, the catalytic activity of the xylan-degrading hydrolase could be easily monitored in routine plate assays and in liquid test measurements at room temperature when produced in recombinant form in E. coli. This tool allows the indirect detection of the intein’s catalytic activity to be used in screenings.
Subjects
Extein
Intein
Picrophilus torridus
Protein splicing
Truncation
Xylanase
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