Orthogonal Synthetic Zippers as Protein Scaffolds

ACS Omega
George P AndersonEllen R Goldman

Abstract

Protein scaffolds have proven useful for co-localization of enzymes, providing control over stoichiometry and leading to higher local enzyme concentrations, which have led to improved product formation. To broaden their usefulness, it is necessary to have a wide choice of building blocks to mix and match for scaffold generation. Ideally, the scaffold building blocks should function at any location within the scaffold and have high affinity interactions with their binding partners. We examined the utility of orthogonal synthetic coiled coils (zippers) as scaffold components. The orthogonal zippers are coiled coil domains that form heterodimers only with their specific partner and not with other zipper domains. Focusing on two orthogonal zipper pairs, we demonstrated that they are able to function on either end or in the middle of a multiblock assembly. Surface plasmon resonance was employed to assess the binding kinetics of zipper pairs placed at the start, middle, or end of a construct. Size-exclusion chromatography was used to demonstrate the ability of a scaffold with two zipper domains to bind their partners simultaneously. We then expanded the study to examine the binding kinetics and cross-reactivities of three additional ...Continue Reading

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Citations

Dec 21, 2018·Nature·Zibo ChenDavid Baker
Jan 14, 2021·Biotechnology and Applied Biochemistry·Surabhi Soni
Jul 24, 2021·International Journal of Biological Macromolecules·Abdallah R IsmailKwang-Hyun Baek
Jan 27, 2019·ACS Chemical Biology·Maja KlausMartin Grininger
May 7, 2020·Bioconjugate Chemistry·Lenne J M LemmensLuc Brunsveld

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Methods Mentioned

BETA
surface
size-exclusion chromatography
surface plasmon resonance
chip
gel filtration
PCR
size-exclusion purification

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