caa a22 4500 605501270 CHVBK 20210128100600.0 cr unu---uuuuu 210128e20150901xx s 000 0 eng 10.1007/s00253-015-6689-8 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6689-8 A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein [Elektronische Daten] [Amélie von der Heyde, Julia Lockhauserbäumer, Charlotte Uetrecht, Skander Elleuche] 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. Springer-Verlag Berlin Heidelberg, 2015 Intein nationallicence Extein nationallicence Truncation nationallicence Protein splicing nationallicence Xylanase nationallicence Picrophilus torridus nationallicence von der Heyde Amélie Hamburg University of Technology (TUHH), Institute of Technical Microbiology, Kasernenstr. 12, 21073, Hamburg, Germany aut Lockhauserbäumer Julia Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Dynamics of viral structures, Martinistrasse 52, 20251, Hamburg, Germany aut Uetrecht Charlotte Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Dynamics of viral structures, Martinistrasse 52, 20251, Hamburg, Germany aut Elleuche Skander Hamburg University of Technology (TUHH), Institute of Technical Microbiology, Kasernenstr. 12, 21073, Hamburg, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/18(2015-09-01), 7613-7624 0175-7598 99:18<7613 2015 99 253 https://doi.org/10.1007/s00253-015-6689-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6689-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- von der Heyde Amélie Hamburg University of Technology (TUHH), Institute of Technical Microbiology, Kasernenstr. 12, 21073, Hamburg, Germany aut NATIONALLICENCE 700 1- Lockhauserbäumer Julia Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Dynamics of viral structures, Martinistrasse 52, 20251, Hamburg, Germany aut NATIONALLICENCE 700 1- Uetrecht Charlotte Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Dynamics of viral structures, Martinistrasse 52, 20251, Hamburg, Germany aut NATIONALLICENCE 700 1- Elleuche Skander Hamburg University of Technology (TUHH), Institute of Technical Microbiology, Kasernenstr. 12, 21073, Hamburg, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/18(2015-09-01), 7613-7624 0175-7598 99:18<7613 2015 99 253