Patent ID: 8288123
Filing Date: 2012-10-16
Classification: A61P,C12N

Abstract:
1. A method of providing a subject polypeptide variant with improved stability compared with a parent subject polypeptide, the method comprising: (a) providing mRNA molecules, each mRNA molecule comprising a nucleotide sequence encoding a subject polypeptide variant and lacking an in-frame stop codon; (b) incubating the mRNA molecules under conditions for ribosome translation of the mRNA molecules to produce encoded subject polypeptide variants, whereby complexes each comprising at least a ribosome, mRNA and encoded subject polypeptide variant are formed; (c) bringing the complexes into contact with a receptor, ligand or specific binding pair member that binds the parent subject polypeptide, and selecting one or more complexes each displaying a subject polypeptide variant able to bind the receptor, ligand or specific binding pair member under the conditions of the selection; wherein dithiothreitol (DTT) is applied as a stability selection pressure during translation in step (b) and continues to be applied during selecting in step (c), and hydrophobic interaction chromatography (HIC) and room temperature are applied simultaneously with DTT as further stability selection pressures during selecting in step (c), or are applied simultaneously with DTT after translation in step (b) and removed prior to bringing the complexes into contact with the receptor, ligand or specific binding pair member in step (c); and (d) determining stability of selected subject polypeptide variant or variants, whereby one or more subject polypeptide variants with improved stability compared with the parent subject polypeptide are obtained.