Cell-free expression of soluble and membrane proteins in an array device for drug screening

Ruba Khnouf, Daniel Olivero, Shouguang Jin, Matthew A Coleman, Z. Hugh Fan

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Enzymes and membrane protein receptors represent almost three-quarters of all current drug targets. As a result, it would be beneficial to have a platform to produce them in a high-throughput format for drug screening. We have developed a miniaturized fluid array device for cell-free protein synthesis, and the device was exploited to produce both soluble and membrane proteins. Two membrane-associated proteins, bacteriorhodopsin and ApoA lipoprotein, were coexpressed in an expression medium in the presence of lipids. Simultaneous expression of ApoA lipoprotein enhanced the solubility of bacteriorhodopsin and would facilitate functional studies. In addition, the device was employed to produce two enzymes, luciferase and β-lactamase, both of which were demonstrated to be compatible with enzyme inhibition assays. β-lactamase, a drug target associated with antibiotic resistance, was further used to show the capability of the device for drug screening. β-Lactamase was synthesized in the 96 units of the device and then assayed by a range of concentrations of four mock drug compounds without harvesting and purification. The inhibitory effects of these compounds on β-lactamase were measured in a parallel format, and the degree in their drug effectiveness agreed well with the data in the literature. This work demonstrated the feasibility of the use of the fluid array device and cell-free protein expression for drug screening, with advantages in less reagent consumption, shorter analysis time, and higher throughput.

Original languageEnglish (US)
Pages (from-to)7021-7026
Number of pages6
JournalAnalytical Chemistry
Volume82
Issue number16
DOIs
StatePublished - Aug 15 2010
Externally publishedYes

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Screening
Membrane Proteins
Pharmaceutical Preparations
Apolipoproteins A
Bacteriorhodopsins
Lipoproteins
Throughput
Enzyme inhibition
Fluids
Enzymes
Luciferases
Purification
Assays
Proteins
Solubility
Anti-Bacterial Agents
Lipids

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Cell-free expression of soluble and membrane proteins in an array device for drug screening. / Khnouf, Ruba; Olivero, Daniel; Jin, Shouguang; Coleman, Matthew A; Fan, Z. Hugh.

In: Analytical Chemistry, Vol. 82, No. 16, 15.08.2010, p. 7021-7026.

Research output: Contribution to journalArticle

Khnouf, Ruba ; Olivero, Daniel ; Jin, Shouguang ; Coleman, Matthew A ; Fan, Z. Hugh. / Cell-free expression of soluble and membrane proteins in an array device for drug screening. In: Analytical Chemistry. 2010 ; Vol. 82, No. 16. pp. 7021-7026.
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