Thermal precipitation fluorescence assay for protein stability screening

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Science

Background:

• High-throughput screening of recombinant proteins requires efficient methods for assessing protein stability.
• Current methods often necessitate complex protein purification steps, limiting their applicability in screening.

Purpose of the Study:

• To introduce a straightforward and reliable assay, termed thermal precipitation fluorescence (TPF), for comparing the thermal stabilities of recombinant proteins.
• To adapt the TPF assay for screening membrane proteins directly from cell lysate supernatants, facilitating high-throughput expression analysis.

Main Methods:

• Recombinant proteins fused with a green fluorescence protein tag were expressed and solubilized with detergent.
• Thermal stability was assessed by applying a heat shock, followed by centrifugation to remove aggregates.
• Remaining soluble protein was quantified using in-gel fluorescence analysis and compared to untreated samples.

Main Results:

• The TPF assay successfully compared thermal stabilities of over 60 recombinant membrane proteins from Escherichia coli.
• The assay was validated in the presence and absence of various detergents.
• No sophisticated protein purification was required, demonstrating the assay's efficiency.

Conclusions:

• The thermal precipitation fluorescence (TPF) technique is suitable for high-throughput expression screening of both membrane and soluble recombinant proteins.
• TPF allows direct assessment of protein stability from cell lysates, simplifying the screening process.
• This method can prioritize target proteins based on thermal stability for large-scale expression and structural studies.