Mutant hydrolase proteins with enhanced kinetics and functional expression
View Patent ↗The invention provides a mutant hydrolase protein with enhanced kinetics and functional expression, as well as polynucleotides encoding the mutant proteins and methods of using the polynucleotides and mutant proteins.
1. A polypeptide comprising a mutant dehalogenase that is capable of catalyzing hydrolytic dehalogenation of an alkylhalide substrate and forming a covalent ester intermediate with the substrate, said mutant dehalogenase having at least 85% sequence identity with the polypeptide of SEQ ID NO: 1; wherein said mutant dehalogenase comprises substitutions at:
(a) one or more positions corresponding to positions in SEQ ID NO: 1 selected from the group consisting of positions 106 and 272; and
(b) positions corresponding to positions 58, 78, 155, 167, 172, 224, and 291 of SEQ ID NO: 1.
2. A fusion polypeptide comprising the polypeptide of claim 1 and a polypeptide of interest.
3. The fusion polypeptide of claim 2 , wherein the polypeptide of interest is at the C-terminus of the fusion polypeptide.
4. The fusion polypeptide of claim 2 , wherein the polypeptide of interest is at the N-terminus of the fusion polypeptide.
5. The fusion polypeptide of claim 2 , further comprising a polypeptide linker linking the polypeptide of claim 1 and the polypeptide of interest.
6. The fusion polypeptide of claim 5 , wherein the linker comprises a protease recognition sequence.