His-MBP tagged DNA endonuclease for facilitated enzyme removal
AssigneeAbClonal Science, Inc.
InventorsZhenyu Zhu, Dapeng Sun, Michaela Shottes
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Abstract
Disclosed is a product and process, wherein one adds a N-terminal Histamine-Maltose Binding Protein (“MBP”) tag to endonucleases, including restriction endonucleases like Hind III, and binds the tagged fusion protein to a solid support, preferably beads, once the enzyme has digested oligonucleotides in solution, in order to arrest further digestion. Preferred beads for binding the tagged enzyme are magnetic beads, which can easily be removed from solution by binding to a support and then removing it, or can be accumulated by magnetic attraction in a particular region. More preferred are magnetic beads bound to iminodiacetic acid or nitrilotriacetic acid.
Claims14
A process of inactivating an endonuclease fusion protein with a tag sequence, following a specified quantity or amount of substrate digestion, comprising:
- providing reaction conditions for a reaction mixture including the fusion protein and a DNA oligomer;
- allowing a reaction to proceed for a specified period or until a specified quantity of the DNA oligomer has been digested;
- and terminating the digestion reaction by binding the tag portion of the fusion protein to a solid support, wherein the tag is an N-terminal tag having the amino acid sequence MKIHHHHHH (SEQ ID NO:3).
Dependencies are inferred from the claim text.