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High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering.
Jason J Lavinder1, Sanjay B Hari, Brandon J Sullivan
1Ohio State Biochemistry Program, Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Journal of the American Chemical Society
|March 19, 2009
Summary
A new high-throughput thermal scanning (HTTS) method uses a hydrophobic dye to rapidly assess protein stability. This cost-effective technique enables the discovery of stable protein variants for various applications.
Area of Science:
- Biochemistry
- Biotechnology
- Protein Engineering
Background:
- Protein stability is crucial for therapeutic, industrial, and research applications.
- Current methods for assessing protein stability lack the necessary throughput and scalability.
- Limited throughput restricts the examination of protein variants for improved stability.
Purpose of the Study:
- To develop a high-throughput, low-cost method for determining protein variant stability.
- To enable the screening of a large number of protein variants efficiently.
- To overcome limitations of existing protein stability assessment techniques.
Main Methods:
- Developed a high-throughput thermal scanning (HTTS) method.
- Utilized a hydrophobic dye (akin to ANS) that fluoresces upon binding to molten globules and thermal denaturation intermediates.
- Employed a real-time PCR machine for analysis of small sample sizes.
- Validated the method using four-helix bundle hydrophobic core variants and TIM barrel variants.
Main Results:
- HTTS provides approximate protein stabilities at high throughput and low cost.
- The method does not require inherent protein properties like enzymatic activity or intrinsic fluorescence.
- Apparent T(M) values from HTTS showed an approximate linear correlation with CD thermal denaturation values.
- Successful application to four-helix bundle and TIM barrel variants.
Conclusions:
- HTTS is an inexpensive, general, and scalable approach for identifying stable protein mutants.
- This method facilitates the search for stable variants of biotechnologically important proteins.
- Provides a new tool for the statistical correlation of protein sequence-stability relationships.
