Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins

TY - JOUR

T1 - Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins

AU - Wright, James N.

AU - Wong, Wan Ling

AU - Harvey, Joseph A.

AU - Garnett, James A.

AU - Itzhaki, Laura S.

AU - Main, Ewan R.G.

PY - 2019/5/7

Y1 - 2019/5/7

N2 - 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. Herein, Wright et al. present an expandable recombinant assembly system that produces scalable protein cages via split intein-mediated native chemical ligation. It uses symmetry designed protein fusions coupled with a driving force of orthogonal split inteins. This combination enables one-pot construction of two-component cages and stepwise assembly of three-component cages.

AB - 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. Herein, Wright et al. present an expandable recombinant assembly system that produces scalable protein cages via split intein-mediated native chemical ligation. It uses symmetry designed protein fusions coupled with a driving force of orthogonal split inteins. This combination enables one-pot construction of two-component cages and stepwise assembly of three-component cages.

KW - directed protein assembly

KW - native chemical ligation

KW - protein conjugation

KW - protein design

KW - protein semi-synthesis

KW - split intein ligation

UR - http://www.scopus.com/inward/record.url?scp=85064914273&partnerID=8YFLogxK

U2 - 10.1016/j.str.2019.02.005

DO - 10.1016/j.str.2019.02.005

M3 - Article

C2 - 30879889

AN - SCOPUS:85064914273

SN - 0969-2126

VL - 27

SP - 776-784.e4

JO - Structure

JF - Structure

IS - 5

ER -