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Scalable geometrically designed protein cages assembled via genetically encoded split inteins

DOI: 10.1016/j.str.2019.02.005 DOI Help

Authors: James N. Wright (Queen Mary, University of London) , Wan Ling Wong (Queen Mary, University of London) , Joseph A. Harvey (Queen Mary, University of London) , James A. Garnett (Queen Mary, University of London) , Laura S. Itzhaki (University of Cambridge) , Ewan R. G. Main (Queen Mary, University of London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Structure

State: Published (Approved)
Published: March 2019

Abstract: Engineering proteins to assemble into user-defined structures is key in their development for biotechnological applications. However, designing generic rather than bespoke solutions is challenging. Here we describe an expandable recombinant assembly system that produces scalable protein cages via split intein-mediated native chemical ligation. Three types of component are used: two complementary oligomeric “half-cage” protein fusions and an extendable monomeric “linker” fusion. All are composed of modular protein domains chosen to fulfill the required geometries, with two orthogonal pairs of split intein halves to drive assembly when mixed. This combination enables both one-pot construction of two-component cages and stepwise assembly of larger three-component scalable cages. To illustrate the system's versatility, trimeric half-cages and linker constructs comprising consensus-designed repeat proteins were ligated in one-pot and stepwise reactions. Under mild conditions, rapid high-yielding ligations were obtained, from which discrete proteins cages were easily purified and shown to form the desired trigonal bipyramidal structures.

Journal Keywords: directed protein assembly; native chemical ligation; protein conjugation; split intein ligation; protein design; protein semi-synthesis

Subject Areas: Biology and Bio-materials

Instruments: B21-High Throughput SAXS

Added On: 21/03/2019 10:43

Discipline Tags:

Biotechnology Genetics Structural biology Engineering & Technology Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)