Development of high throughput methodologies to isolate stabilized mutants and to identify interfacial residues

Abstract

When eukaryotic or unstable proteins are overexpressed in bacteria, these often form insoluble aggregates called inclusion bodies (IB). Refolding the proteins from inclusion bodies can be challenging, resulting in low yields. Stable variants of proteins with increased half-life, stability and efficacy are also required by pharmaceutical and biotechnology sectors. Mutagenesis is used to generate protein variants with improved biophysical properties such as solubility and activity. The properties of several vaccines and biological products have been enhanced using mutagenesis. With the advancement of high-throughput mutagenesis and next generation sequencing techniques, a large number of mutants can be generated and evolved for desired activity in a short duration. Display techniques, like yeast surface display and phage display have been used as tools to find protein variants with improved biophysical and biochemical properties. The protein of interest is fused to a surface protein of yeast or phage, which results surface display of the protein. Using display techniques, a protein with desired activity or stability can be obtained after several rounds of selection and mutagenesis. However, an efficient, high-throughput methodology is not currently available to measure the activity, solubility and stability of each variant. It has bee shown in a few cases, that the amount of protein secreted by the yeast cells is correlated with the in vivo solubility and thermal stability of the protein, though contradictory results are also present. Studies which showed a good correlation typically involved proteins that have low stability. No such correlation has been reported for proteins with high stability. It has also been suggested that if the stability of a protein crosses a threshold, its expression does not increase linearly with increase in stability, and it is therefore difficult in such cases to distinguish stable mutants from the less stable ones using only expression as the criterion.