Journal: Proc Natl Acad Sci U S A / Year: 2023 Title: Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains. Authors: Jing Yang John Wang / Alena Khmelinskaia / William Sheffler / Marcos C Miranda / Aleksandar Antanasijevic / Andrew J Borst / Susana V Torres / Chelsea Shu / Yang Hsia / Una Nattermann / ...Authors: Jing Yang John Wang / Alena Khmelinskaia / William Sheffler / Marcos C Miranda / Aleksandar Antanasijevic / Andrew J Borst / Susana V Torres / Chelsea Shu / Yang Hsia / Una Nattermann / Daniel Ellis / Carl Walkey / Maggie Ahlrichs / Sidney Chan / Alex Kang / Hannah Nguyen / Claire Sydeman / Banumathi Sankaran / Mengyu Wu / Asim K Bera / Lauren Carter / Brooke Fiala / Michael Murphy / David Baker / Andrew B Ward / Neil P King / Abstract: Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed ...Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.
History
Deposition
Nov 29, 2022
Deposition site: RCSB / Processing site: RCSB
Revision 1.0
Mar 22, 2023
Provider: repository / Type: Initial release
Revision 1.1
May 22, 2024
Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Mass: 36866.043 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.64 Å3/Da / Density % sol: 53.33 %
Crystal grow
Temperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7 Details: 0.2 M magnesium chloride, 0.1 M Tris, pH 7.0, 10% w/v PEG8000
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Data collection
Diffraction
Mean temperature: 100 K / Serial crystal experiment: N
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