Mutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag

Frontiers in Bioengineering and Biotechnology

Abdallah M. Abdallah²  Jan-Willem de Gier⁴  Wendy Dontje⁵  Corinne M. ten Hagen-Jongman⁶  Joen Luirink⁶  Wouter S. P. Jong⁶  Wilbert Bitter⁷  David Vikström⁸ 

[1] Abera Bioscience AB, Solna, Sweden;Bioscience Core Laboratory, King Abdullah University of Science and Technology (KAUST), Jeddah, Saudi Arabia;Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha, Qatar;Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, Stockholm, Sweden;Department of Clinical Immunology and Rheumatology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands;Department of Molecular Microbiology, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Vrije Universiteit, Amsterdam, Netherlands;Medical Microbiology and Infection Control, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit, Amsterdam, Netherlands;Xbrane Biopharma AB, Solna, Sweden;

关键词: inclusion bodies;    fusion tag;    insoluble expression;    protein aggregation;    heterologous protein production;    signal peptide;   

DOI  :  10.3389/fbioe.2019.00442

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown