Thermo-stable proteins are of great industrial importance. Say, I want to increase the stability of a protein by raising its optimal temp from 50 to 70°C. It is relatively easy to create a mutation library by error-prone PCR or DNA shuffling. But the challenge is how to find right hits by screening thousands of mutants in a high throughput way. Any ideas?
It depends on the enzyme in which you are interested. If its direct or even indirect activity (by coupling to a second enzyme) can be used to catalyze a reaction which can use a tagged substrate ((fluorescence or a chromogenic) such that once the substrate is converted to the product, it becomes insoluble, you ca use easily do it on a chip.
Differential scanning fluorimetry is a technique that allows you to measure the thermal stability of protein samples in a plate-based format. You need to have a sample of purified protein in each well.
As Adam Shapiro pointed out thermal shift assay is the way to go. Assay is straight forward when you have purified proteins. In the 384-well fromat assay we perfom routinely, 10 micro-gram protein will be enough to get an answer.
If you can over-express your enzyme, then you can use many techniques for checking the enzyme thermostability. For high-throughput format, I think one of the best available methods is Thermofluor, in which you use a fluorescent dye, Sypro orange, for monitoring the enzyme unfolding in a temperature gradient from 20-99°C. 20ul of 1mg/ml enzyme is enough for this assay.
The question is that I might have thousands of bacterial colonies that overexpress the mutated protein. Do I need to extract and purify those proteins from each of those colonies before I do the thermal shift test?
It depends on how good is the expression. If your mutants are over-expressed in high content and you have comparatively much lower protein background, maybe you can omit protein purification and check stability of your mutants in cell free extract
I think if your protein is an enzyme you can high throughput screen using fluorogenic substrate: after growth of your recombinant bacteria in 384-well microplates with LB+antibiotic+ inducer (overnight/37) you lyse it (using lysis solution) and you add the substrate solution and then you incubate in high temperature, so only thermo-resistant enzymatic activities will be detected during reading fluorescence (using special microplate reader for fluorescence)
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology