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Licensed Unlicensed Requires Authentication Published by De Gruyter September 14, 2016

Development of a screening system for inteins active in protein splicing based on intein insertion into the LacZα-peptide

  • Maximilian Neugebauer , Jana K. Böcker , Julian C.J. Matern , Shmuel Pietrokovski and Henning D. Mootz EMAIL logo
From the journal Biological Chemistry


Protein splicing by inteins has found diverse applications in biotechnology, protein chemistry and chemical biology. Inteins display a wide range of efficiencies and rates unpredictable from their amino acid sequences. Here, we identified positions T22S and S35 in the LacZα peptide as intein insertion sites that strictly require protein splicing, in contrast to cleavage side-reactions, to allow for complementation of β-galactosidase activity. Both the cis-variant of the M86 mutant of the Ssp DnaB intein and a split form undergoing protein trans-splicing gave rise to formation of blue colonies in the β-galactosidase read-out. Furthermore, we report the two novel, naturally split VidaL T4Lh-1 and VidaL UvsX-2 inteins whose N-terminal fragments consist of only 15 and 16 amino acids, respectively. Initial biochemical characterization with the LacZα host system of these inteins further underlines its utility. Finally, we used the LacZα host system to rapidly identify amino acid substitutions from a small randomized library at the structurally conserved intein position 2 next to the catalytic center, that are tolerated for protein splicing activity of the M86 intein. These findings demonstrate the potential of the system for initial testing and directed evolution of inteins.


Financial support by the DFG (grant MO1073/3-2) is gratefully acknowledged. We thank Konrad Büssow (Max Planck Institute for Molecular Genetics, Berlin, Germany) for the kind gift of the pQLinkH plasmid, which was provided through Addgene.


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Supplemental Material:

The online version of this article (DOI: 10.1515/hsz-2016-0229) offers supplementary material, available to authorized users.

Received: 2016-6-10
Accepted: 2016-8-23
Published Online: 2016-9-14
Published in Print: 2017-1-1

©2017 Walter de Gruyter GmbH, Berlin/Boston

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