Engineering an AB5 Protein Carrier

Scientific Reports
Bruce R Lichtenstein, Birte Höcker

Abstract

The promise of biologic therapeutics is hindered by the challenge to deliver their activity to biochemically relevant sites within diseased cells. The favourable application of the natural protein carriers of the AB5 toxin family to this challenge has been restricted owing to still unresolved requirements for assembling non-native cargo into carrier complexes. Here, we clarify the properties of fusion peptides which allow co-assembly of a selected fluorescent protein cargo with the non-toxic B subunit of a heat-labile enterotoxin. We establish the influence of sequence length, sequence identity and secondary structure of these linking domains on the assembly and disassembly of the complexes. Through our engineering framework we identify several non-native, reduced length fusion sequences that robustly assemble with the native carriers, maintain their ability to deliver protein cargo to cells, and demonstrate substantially refined in vitro properties. Constructs based upon these sequences should prove directly applicable to a variety of protein delivery challenges, and the described design framework should find immediate application to other members of the AB5 protein carrier family.

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Citations

Aug 29, 2019·Frontiers in Cellular and Infection Microbiology·Qiangde DuanGuoqiang Zhu

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Methods Mentioned

BETA
tandem affinity purification
gel filtration
confocal microscopy
affinity purification
Fluorescence

Software Mentioned

Fiji
Affinity Designer
PyCORN

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