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dc.contributor.authorJong, Wouter S.P.
dc.contributor.authorten Hagen-Jongman, Corinne M.
dc.contributor.authorVikström, David
dc.contributor.authorDontje, Wendy
dc.contributor.authorAbdallah, Abdallah
dc.contributor.authorde Gier, Jan Willem
dc.contributor.authorBitter, Wilbert
dc.contributor.authorLuirink, Joen
dc.identifier.citationJong, W. S. P., ten Hagen-Jongman, C. M., Vikström, D., Dontje, W., Abdallah, A. M., de Gier, J.-W., … Luirink, J. (2020). Mutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag. Frontiers in Bioengineering and Biotechnology, 7. doi:10.3389/fbioe.2019.00442
dc.description.abstractWhereas, bacterial inclusion bodies (IBs) for long were regarded as undesirable aggregates emerging during recombinant protein production, they currently receive attention as promising nanoparticulate biomaterials with diverse applications in biotechnology and biomedicine. We previously identified ssTorA, a signal sequence that normally directs protein export via the Tat pathway in E. coli, as a tag that induces the accumulation of fused proteins into IBs under overexpression conditions. Here, we used targeted mutagenesis to identify features and motifs being either critical or dispensable for IB formation. We found that IB formation is neither related to the function of ssTorA as a Tat-signal sequence nor is it a general feature of this family of signal sequences. IB formation was inhibited by co-overexpression of ssTorA binding chaperones TorD and DnaK and by amino acid substitutions that affect the propensity of ssTorA to form an α-helix. Systematic deletion experiments identified a minimal region of ssTorA required for IB formation in the center of the signal sequence. Unbiased genetic screening of a library of randomly mutagenized ssTorA sequences for reduced aggregation properties allowed us to pinpoint residues that are critical to sustain insoluble expression. Together, the data point to possible mechanisms for the aggregation of ssTorA fusions. Additionally, they led to the design of a tag with superior IB-formation properties compared to the original ssTorA sequence.
dc.description.sponsorshipWe would like to thank Elise Riesebos, Rosa Luirink, and Erik van Duijvenvoorde for excellent technical assistance. Olivier Fayet (LMGM, Toulouse, France) and Bernard Connolly (University of Newcastle, Newcastle upon Tyne, UK) are gratefully acknowledged for their contribution of plasmids and reagents.
dc.publisherFrontiers Media SA
dc.rightsArchived with thanks to Frontiers in Bioengineering and Biotechnology
dc.rightsThis file is an open access version redistributed from:
dc.titleMutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Division
dc.contributor.departmentBioscience Core Lab
dc.contributor.departmentComputational Bioscience Research Center (CBRC)
dc.contributor.departmentComputer, Electrical and Mathematical Sciences and Engineering (CEMSE) Division
dc.contributor.departmentNGS, qPCR and Single Cell Genomics
dc.contributor.departmentSanger and Third Generation Sequencing
dc.identifier.journalFrontiers in Bioengineering and Biotechnology
dc.eprint.versionPublisher's Version/PDF
dc.contributor.institutionAbera Bioscience AB, Solna, Sweden
dc.contributor.institutionDepartment of Molecular Microbiology, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Vrije Universiteit, Amsterdam, Netherlands
dc.contributor.institutionXbrane Biopharma AB, Solna, Sweden
dc.contributor.institutionDepartment of Clinical Immunology and Rheumatology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands
dc.contributor.institutionDepartment of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha, Qatar
dc.contributor.institutionDepartment of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, Stockholm, Sweden
dc.contributor.institutionMedical Microbiology and Infection Control, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit, Amsterdam, Netherlands
kaust.personAbdallah, Abdallah

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