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 language | English (US) |
|---|---|
| Pages (from-to) | 7021-7026 |
| Number of pages | 6 |
| Journal | Analytical Chemistry |
| Volume | 82 |
| Issue number | 16 |
| DOIs | |
| State | Published - Aug 15 2010 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Cell-free expression of soluble and membrane proteins in an array device for drug screening
AU - Khnouf, Ruba
AU - Olivero, Daniel
AU - Jin, Shouguang
AU - Coleman, Matthew A
AU - Fan, Z. Hugh
PY - 2010/8/15
Y1 - 2010/8/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=77955647887&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77955647887&partnerID=8YFLogxK
U2 - 10.1021/ac1015479
DO - 10.1021/ac1015479
M3 - Article
C2 - 20666430
AN - SCOPUS:77955647887
VL - 82
SP - 7021
EP - 7026
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 16
ER -