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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

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Published on: January 6, 2015

Automated, high-throughput platform for protein solubility screening using a split-GFP system.

Pawel Listwan¹, Thomas C Terwilliger, Geoffrey S Waldo

¹Bioscience Division, MS-M888, Los Alamos National Laboratory, Bikini Atoll Rd, SM30, Los Alamos, NM 87545, USA.

Journal of Structural and Functional Genomics

|November 29, 2008

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Researchers developed an automated platform using split-green fluorescent protein (GFP) tags to efficiently identify stable protein domains. This technology overcomes bottlenecks in protein production for structural and functional studies.

Area of Science:

Biochemistry
Molecular Biology
Structural Biology

Background:

Producing soluble and stable proteins is crucial for structural genomics and biochemical research but often challenging for difficult proteins.
Advances in protein stability technologies, including protein domain trapping and solubility mutations, aim to address these challenges.
Split-green fluorescent protein (GFP) tags offer a powerful method for detecting stable protein domains.

Purpose of the Study:

To develop and validate an automated, high-throughput liquid-handling platform for protein expression and analysis.
To optimize protocols for bacterial culture, lysis, and protein extraction within a 96-well plate format.
To enable rapid determination of optimal protein domains from various genetic sources.

Main Methods:

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Utilized split-GFP protein tags, where a 15-amino acid GFP fragment (216-228) is fused to target proteins.
Employed an engineered GFP 1-10 "detector" fragment (1-215) for in vivo and in vitro detection of tagged proteins via fluorescence.
Adapted manual benchtop processes to a high-throughput, automated liquid-handling system for protein assays.

Main Results:

Successfully optimized bacterial culture, lysis, and protein extraction protocols for a 96-well plate format.
Validated the automated system for assessing soluble and insoluble protein levels.
Demonstrated the utility of the split-GFP system for identifying stable protein domains.

Conclusions:

The automated liquid-handling platform significantly enhances the efficiency of identifying soluble and stable protein domains.
Split-GFP technology, when integrated into high-throughput platforms, provides a robust solution for protein expression challenges.
This approach accelerates the discovery of suitable protein reagents for functional and structural studies.