Methods for selecting enzymes having protease activity
AssigneeNovozymes A/S
InventorsRobert Blazej, Nicholas Toriello, Charles Emrich
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Abstract
Provided herein are systems and components thereof for improving protease activity. The systems make use of an emulsion for in vitro compartmentalization of a library of synthetic compounds, each compound having a gene linked to a protease substrate and selectable marker. Expressed enzymes with greater protease activity will preferentially hydrolyze the protease substrate, whereas enzymes with less protease activity will leave the substrate intact. Removal of the non-hydrolyzed compounds provides an enriched gene library encoding for more active protease variants. Also described are synthetic compounds and emulsions which can be used in the methods.
Claims20
A method of selecting for a polypeptide having protease activity, the method comprising:
- suspending a plurality of synthetic compounds in an aqueous phase, wherein the synthetic compounds individually comprise:
- (a) a polynucleotide encoding for a polypeptide;
- (b) a protease substrate linked to said polynucleotide;
- and
- (c) a selectable marker linked to said polynucleotide;
- wherein the aqueous phase comprises components for expression of the polypeptide;
- (ii) forming a water-in-oil emulsion with the aqueous phase, wherein the synthetic compounds are compartmentalized in aqueous droplets of the emulsion;
- (iii) expressing the polypeptides within the aqueous droplets of the emulsion, wherein a polypeptide with protease activity in an aqueous droplet hydrolyzes the protease substrate in that droplet;
- and
- (iv) separating the synthetic compounds to recover synthetic compounds comprising the protease substrate and/or synthetic compounds wherein the protease substrate has been hydrolyzed.
A synthetic compound comprising:
- (a) a polynucleotide encoding for a polypeptide;
- (b) a protease substrate linked to said polynucleotide;
- and
- (c) a selectable marker linked to said polynucleotide.
Dependencies are inferred from the claim text.