Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification

James H. Wade, Joshua D. Jones, Ivan L. Lenov, Colleen M. Riordan, Stephen G. Sligar, Ryan C. Bailey

Research output: Contribution to journalArticle

Abstract

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

Original languageEnglish (US)
Pages (from-to)2951-2959
Number of pages9
JournalLab on a chip
Volume17
Issue number17
DOIs
StatePublished - Jan 1 2017

Fingerprint

Microfluidics
Purification
Membrane Proteins
Proteins
Membranes
Miniaturization
Biochemistry
Phospholipids
Cell culture
Solubility
Cytochrome P-450 Enzyme System
Catalytic Domain
Screening
Lipids
Mass transfer
Cell Culture Techniques
Chemical analysis

ASJC Scopus subject areas

  • Bioengineering
  • Biochemistry
  • Chemistry(all)
  • Biomedical Engineering

Cite this

Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification. / Wade, James H.; Jones, Joshua D.; Lenov, Ivan L.; Riordan, Colleen M.; Sligar, Stephen G.; Bailey, Ryan C.

In: Lab on a chip, Vol. 17, No. 17, 01.01.2017, p. 2951-2959.

Research output: Contribution to journalArticle

Wade, James H. ; Jones, Joshua D. ; Lenov, Ivan L. ; Riordan, Colleen M. ; Sligar, Stephen G. ; Bailey, Ryan C. / Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification. In: Lab on a chip. 2017 ; Vol. 17, No. 17. pp. 2951-2959.
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