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

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• 作者：Amélie von der Heyde ; Julia Lockhauserb?umer…
• 关键词：Intein ; Extein ; Truncation ; Protein splicing ; Xylanase ; Picrophilus torridus
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2015
• 出版时间：September 2015
• 年：2015
• 卷：99
• 期：18
• 页码：7613-7624
• 全文大小：1,804 KB
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  Shen B, Sun X, Zuo X, Shilling T, Apg
• 作者单位：Amélie von der Heyde (1)
  Julia Lockhauserb?umer (2)
  Charlotte Uetrecht (2) (3)
  Skander Elleuche (1)
  
  1. Hamburg University of Technology (TUHH), Institute of Technical Microbiology, Kasernenstr. 12, 21073, Hamburg, Germany
  2. Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Dynamics of viral structures, Martinistrasse 52, 20251, Hamburg, Germany
  3. European XFEL GmbH, Sample Environment Group, Notkestrasse 85, 22607, Hamburg, Germany
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

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 ? 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. Keywords Intein Extein Truncation Protein splicing Xylanase Picrophilus torridus