T4 DNA ligase variants with increased resistance to salt
A number of T4 DNA ligase mutants exhibiting enhanced ligation activity in the presence of high salt concentrations compared to the wild-type ligase were engineered, characterized, and selected via gel electrophoresis of ligation products from a standard ligation assay. Ligase catalyzes the formation of phosphodiester bonds between the 5′ and 3′ ends of complementary cohesive ends or blunt ends of duplex DNA, a process that is vital to numerous molecular biology processes including cloning and sequencing.
1. A mutant T4 DNA ligase having 95% sequence identity to SEQ ID NO:4 and comprising the amino acid substitution E88K.
2. A mutant T4 DNA ligase having 99% sequence identity to SEQ ID NO:4 and comprising the amino acid substitution E88K.
3. A process of conducting polynucleotide ligation between two different polynucleotides or by ligating the 5′ and 3′ end of a single polynucleotide to generate a circular polynucleotide, wherein the polynucleotides have blunt ends or cohesive ends, comprising: providing a ligation mixture including the polynucleotide(s) to be ligated and the mutant T4 DNA ligase of claim 1 with a concentration of salt in the ligation mixture of greater than or equal to 250 mM; and placing said ligation mixture at a temperature wherein ligation takes place.
4. The process of claim 3 wherein the salt is NaCl.
5. The process of claim 3 wherein the ligation reaction mixture includes Tris-HCl, MgCl 2 , ATP, dithiothreitol and water.
6. A process of conducting polynucleotide ligation between two different polynucleotides or by ligating the 5′ and 3′ end of a single polynucleotide to generate a circular polynucleotide, wherein the polynucleotides have blunt ends or cohesive ends, comprising: providing a ligation mixture including the polynucleotide(s) to be ligated and the mutant T4 DNA ligase of claim 2 with a concentration of salt in the ligation mixture of greater than or equal to 250 mM; and placing said ligation mixture at a temperature wherein ligation takes place.
7. The process of claim 6 wherein the salt is NaCl.
8. The process of claim 6 wherein the ligation reaction mixture includes Tris-HCl, MgCl 2 , ATP, dithiothreitol and water.
9. The mutant T4 DNA ligase of claim 1 , having the amino acid sequence of SEQ ID NO: 4, but not including the 6-membered histidine tag at its C-terminus and the seven immediately preceding Glycine and Serine amino acids.