T7 DNA ligase variants with increased ligation activity
The invention includes a mutant T7 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T7 DNA ligase. The mutant T7 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary. The preferred mutant T7 DNA ligase has at least one of the following mutations: E63K (SEQ ID NO:4), K73E (SEQ ID NO:6), K137E (SEQ ID NO:7), K174E (SEQ ID NO:9), E182K (SEQ ID NO:11), K210E (SEQ ID NO: 13), E243K (SEQ ID NO:15), D245R (SEQ ID NO: 17), E268K (SEQ ID NO:19), E272K (SEQ ID NO:21), E289K (SEQ ID NO:23), K295E (SEQ ID NO:25) and D336R (SEQ ID NO:27).
1 . A mutant T7 DNA ligase or a biologically active fragment thereof, comprising the mutation E63K (SEQ ID NO:4), but wherein amino acids in SEQ ID NO:4 other than 63K can have a conservative mutation, but only to the extent that there remains at least 90% sequence identity to SEQ ID NO:4.
2 . A mutant T7 DNA ligase or a biologically active fragment thereof, comprising the mutation E63K (SEQ ID NO:4), but wherein amino acids in SEQ ID NO:4 other than 63K can have a conservative mutation, but only to the extent that there remains at least 95% sequence identity to SEQ ID NO:4.
3 . A mutant T7 DNA ligase of claim 2 wherein amino acids in SEQ ID NO:4 other than 63K can have a conservative mutation, but only to the extent that there remains at least 99% sequence identity to SEQ ID NO:4.
4 . A mutant T7 DNA ligase having the amino acid sequence of SEQ ID NO:4.