Methods of screening proteins

A method of isolating at least one polynucleotide encoding a stable mutant binding protein, said method comprising

• a) culturing host cells under conditions suitable for protein expression, said host cells comprising collectively a set of expression vectors, wherein said set of expression vectors comprises collectively polynucleotides encoding at least one member of a protein library of mutant binding proteins;
• b) contacting said cultured host cells with at least one ligand;
• c) detecting differences in an interaction among individual host cells with said ligand, said differences in interaction of said individual host cells with said ligand indicating differences in said mutant protein stability and in the ability of said at least one expressed mutant binding protein within said individual host cells to interact with said ligand;
• d) separating said individual host cells displaying the desired interaction with said ligand from said individual host cells that do not display the desired interaction with said ligand;
• and
• e) separating the expression vector from said individual host cells displaying said desired interaction, said expression vector comprising the polynucleotide encoding said stable mutant binding protein capable of specifically binding said ligand.

A method of generating at least one stable mutant binding protein with desired properties, said method comprising

• a) culturing host cells under conditions suitable for protein expression, said host cells comprising collectively a set of expression vectors, wherein said set of expression vectors comprises collectively polynucleotides encoding at least one member of a protein library of mutant binding proteins;
• b) contacting said cultured host cells with at least one ligand;
• c) detecting differences in an interaction among individual host cells with said ligand, said differences in interaction of said individual host cells with said ligand indicating differences in said mutant protein stability and in the ability of said at least one expressed mutant binding protein within said individual host cells to interact with said ligand;
• d) separating said individual host cells displaying the desired interaction with said ligand from said individual host cells that do not display the desired interaction with said ligand;
• e) isolating the expression vector from said individual host cells displaying said desired interaction, said expression vector comprising a polynucleotide encoding a stable mutant binding protein capable of specifically binding said ligand;
• f) generating a subsequent mutant protein library of said stable mutant binding protein in (e), said generation of said subsequent mutant library comprising targeted random mutagenesis of said polynucleotide encoding said first stable mutant binding protein from (e) to produce said subsequent mutant protein library;
• and
• g) repeating (a) through (e) on said subsequent mutant protein library either to obtain additional subsequent mutant protein library or libraries or to obtain a polynucleotide encoding said stable mutant protein with said desired properties;
• said additional subsequent library or libraries being subjected to (a) through (e) until said polynucleotide encoding said stable mutant protein with said desired properties is obtained.