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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
Fluorescent probe for high-throughput screening of membrane protein expression
A E Backmark1, N Olivier, A Snijder
1Discovery Sciences, Reagents and Assay Development, AstraZeneca Pharmaceuticals, Mölndal, Sweden.
Protein Science : a Publication of the Protein Society
|June 19, 2013
Summary
A new fluorescent probe method using NTA technology enhances protein screening by overcoming limitations of traditional GFP fusion proteins. This sensitive technique accurately assesses protein production and stability in crude mixtures.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Protein variant screening requires robust detection methods for levels and stability in complex mixtures.
- Fluorescence-detection size-exclusion chromatography (FSEC) with green fluorescent protein (GFP) fusions is common but has drawbacks like false positives and aggregation.
- Limitations of GFP fusions include reduced yields and issues post-cleavage, necessitating alternative screening strategies.
Purpose of the Study:
- To develop and validate a novel FSEC screening strategy using a fluorescent multivalent NTA probe for polyhistidine-tagged proteins.
- To overcome the disadvantages associated with GFP fusion proteins in protein variant screening.
- To provide a sensitive and quantitative method for assessing protein production and stability.
Main Methods:
- A FSEC screening strategy utilizing a fluorescent multivalent NTA probe that binds to polyhistidine-tags on target proteins.
- Application of the NTA probe method to screen protein variants in crude detergent extracts.
- Utilizing suspension-adapted HEK293-6E cells for transient protein production and analysis.
Main Results:
- The NTA probe method successfully identified domain boundaries of the human G-protein coupled adenosine A2a receptor from crude extracts.
- Efficiently identified well-expressing clones of MraY, a bacterial infection target, from a library of 24 orthologs.
- Demonstrated high sensitivity, detecting target proteins at expression levels as low as 0.02 mg/L in crude lysate with minimal cell culture requirements.
Conclusions:
- The fluorescent NTA probe offers a superior alternative to GFP fusions for FSEC-based protein screening.
- This method provides accurate qualitative and quantitative parameters for ranking protein production.
- The probe is a highly sensitive tool for detecting target proteins in crude lysates, enabling efficient screening of protein variants and identification of optimal expression constructs.
