Enzyme/tag binding and detection system
A hybrid polypeptide composed of an identification peptide and a desired functional protein are produced by recombinant DNA techniques. A DNA expression vector is constructed that includes segments of DNA coding for the identification peptide and the desired functional protein. The identification peptide consists of a specific sequence of amino acids that has the desired affinity for a non-antibody capture protein. This peptide maybe linked to either the amino or carboxyl terminal of the functional protein. The identification peptide may or may not contain a sequence of amino acids that can be fragmented by sequence specific proteases or chemical agents to yield the native protein. The hybrid polypeptide produced by either cell or cell-free based expression systems is now suitable for further processing. This fusion protein can be purified by affinity chromatographic techniques using an immobilized non-antibody capture protein that has the desired affinity for the identification peptide. Alternately the capture protein may contain a label that will allow for the tracking of the functional protein in the system being studied.
1 . A method for affinity purification of a recombinant fusion peptide, said method comprising the steps of:
(a) forming a fusion peptide comprising a tag peptide sequence covalently attached to a polypeptide sequence, the tag sequence comprising a substrate or inhibitor for a serine protease enzyme;
(b) providing an affinity matrix comprising the serine protease enzyme attached to a solid support. the enzyme being modified such that it binds to the tag sequence but is incapable of proteolytically fragmenting the tag sequence;
(c) contacting the fusion peptide with the matrix whereby a complex is formed between the enzyme and the fusion peptide;
(d) eluting non-complexed peptides to separate non-complexed peptides from the complexed fusion peptide; and
(e) eluting the fusion peptide from the enzyme. thereby purifying the fusion peptide.
2 . (canceled)
3 . (canceled)
4 . (canceled)
5 . The method of claim 1 wherein the enzyme is a modified trypsin and the tag sequence comprises the active site sequence of a trypsin inhibitor.
6 . (canceled)
7 . The method of claim 1 wherein the enzyme is a modified enterokinase and the tag sequence comprises the amino terminal sequence of trypsinogen.
8 . (canceled)
9 . The method of claim 1 wherein the tag peptide sequence comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; SEQ ID NO: 13.
10 . (canceled)