Mutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag
Type
ArticleAuthors
Jong, Wouter S.P.ten Hagen-Jongman, Corinne M.
Vikström, David
Dontje, Wendy
Abdallah, Abdallah
de Gier, Jan Willem
Bitter, Wilbert
Luirink, Joen
KAUST Department
Biological and Environmental Sciences and Engineering (BESE) DivisionBioscience Core Lab
Computational Bioscience Research Center (CBRC)
Computer, Electrical and Mathematical Sciences and Engineering (CEMSE) Division
NGS, qPCR and Single Cell Genomics
Sanger and Third Generation Sequencing
Date
2020-01-10Submitted Date
2019-09-26Permanent link to this record
http://hdl.handle.net/10754/661419
Metadata
Show full item recordAbstract
Whereas, 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.Citation
Jong, 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.2019.00442Sponsors
We 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.Publisher
Frontiers Media SAAdditional Links
https://www.frontiersin.org/article/10.3389/fbioe.2019.00442/fullhttps://www.frontiersin.org/articles/10.3389/fbioe.2019.00442/pdf
https://www.frontiersin.org/articles/10.3389/fbioe.2019.00442/pdf
ae974a485f413a2113503eed53cd6c53
10.3389/fbioe.2019.00442
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Except where otherwise noted, this item's license is described as Archived with thanks to Frontiers in Bioengineering and Biotechnology