Hot CoFi Blot: A High-Throughput Colony-Based Screen for Identifying More Thermally Stable Protein Variants

Highly soluble and stable proteins are desirable for many different applications, from basic science to reaching a cancer patient in the form of a biological drug. For X-ray crystallography-where production of a protein crystal might take weeks and even months-a stable protein sample of high purity and concentration can greatly increase the chances of producing a well-diffracting crystal. For a patient receiving a specific protein drug, its safety, efficacy, and even cost are factors affected by its solubility and stability. Increased protein expression and protein stability can be achieved by randomly altering the coding sequence. As the number of mutants generated might be overwhelming, a powerful protein expression and stability screen is required. In this chapter, we describe a colony filtration technology, which allows us to screen random mutagenesis libraries for increased thermal stability-the Hot CoFi blot. We share how to create the random mutagenesis library, how to perform the Hot CoFi blot, and how to identify more thermally stable clones. We use the Tobacco Etch Virus protease as a target to exemplify the procedure.