T4 DNA ligase variants with increased ligation efficiency
The invention includes a mutant T4 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T4 DNA ligase. The mutant T4 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary.
1. A mutant T4 DNA ligase amino acid sequence having only one of the following the amino acid sequences and mutations: E271K in the amino acid sequence of SEQ ID NO: 6, E419K in the amino acid sequence of SEQ ID NO: 14, D452R in the amino acid sequence of SEQ ID NO: 22, and further, wherein each said mutant T4 DNA ligase amino acid sequence does not include the a 6-membered histidine tag at its C-terminus, or a sequence GLGSGSSG preceding the 6-membered histidine tag, and wherein said mutant T4 DNA ligase has greater ligase activity at lower concentrations as compared to a wild type T4 ligase without said mutations.
2. A mutant T4 DNA ligase amino acid sequence of claim 1 having the mutation E271K in the amino acid sequence of SEQ ID NO: 6.
3. A mutant T4 DNA ligase amino acid sequence of claim 1 having the mutation E419K in the amino acid sequence of SEQ ID NO: 14.
4. A mutant T4 DNA ligase amino acid sequence of claim 1 having the mutation D452R in the amino acid sequence of SEQ ID NO: 22.
5. A polynucleotide encoding the amino acid sequence of the mutant T4 DNA ligase of claim 1 .
6. A vector comprising the polynucleotide of claim 5 .
7. A cell transformed with the vector of claim 6 .
8. A process of conducting polynucleotide ligation between different polynucleotides by ligating the 5′ and 3′ ends of polynucleotides to generate a circular polynucleotide, wherein the polynucleotides have blunt ends or cohesive ends, comprising:
providing a ligation mixture including the polynucleotides to be ligated and the mutant T4 DNA ligase of claim 1 ; and placing said ligation mixture at temperatures wherein ligation takes place, thereby generating said circular polynucleotide.
9. A process of conducting polynucleotide ligation between different polynucleotides by ligating the 5′ and 3′ ends of polynucleotides to generate a circular polynucleotide, wherein the polynucleotides have blunt ends or cohesive ends, comprising:
providing a ligation reaction mixture including a buffer, the polynucleotides to be ligated and the mutant T4 DNA ligase of claim 1 ; and placing said ligation reaction mixture under temperature conditions suitable for ligation, thereby generating said circular polynucleotide.
10. The process of claim 9 wherein the ligation reaction mixture comprises Tris-HCl, MgCl2, ATP, dithiothreitol and water.