Short enzyme donor fragment
View Patent ↗Short enzyme donor fragments of β-galactosidase are provided of not more than 40 amino acids, where the short fragments are used as a label and may be substituted with a wide variety of organic compounds, particularly polypeptides having independent functional activity. The enzyme donor finds use in competitive and non-competitive assays, monitoring intracellular events, or other processes where a sensitive non-interfering label is desired.
1. In a cell, an enzyme donor for complementation with an enzyme acceptor fragment of E. coli β-galactosidase, said enzyme donor having from 37 to 40 amino acids of the N-terminal proximal β-galactosidase sequence as a fusion protein linked to a polypeptide of interest.
2. An enzyme donor according to claim 1 , wherein said amino acids have the sequence SEQ ID:NO 1, with not more than a total of three additional amino acids at the termini.
3. An enzyme donor according to claim 2 where the fragment of β-galactosidase is SEQ ID:NO 1.
4. An enzyme donor for complementation with an enzyme acceptor fragment of E. coli β-galactosidase having from 37 to 40 amino acids of the N-terminal proximal β-galactosidase sequence joined by an amino acid linker of from about 1-10 amino acids to a polypeptide of other than the succeeding naturally occurring β-galactosidase sequence of said enzyme donor.
5. An enzyme donor according to claim 4 , wherein said amino acids have the sequence SEQ ID:NO 1, with not more than a total of three additional amino acids at the termini.
6. An enzyme donor according to claim 5 , where the fragment of β-galactosidase is SEQ ID:NO 1.
7. A method for performing an assay intracellularly using enzyme fragment complementation employing a fusion protein comprising a fragments of an E. coli β-galactosidase having from 37 to 40 amino acids of the N-terminal proximal β-galactosidase sequence joined to a polypeptide of interest, wherein the cells employed express said fusion protein, said method comprising:
expressing said fusion protein in said cells; and
determining the presence of said fusion protein by contacting said fusion protein with an enzyme acceptor and a fluorescent or chemiluminescent β-galactosidase substrate.
8. A method according to claim 7 , wherein said enzyme donor is joined to a functional polypeptide expressed in said cells and other than the succeeding naturally occurring β-galactosidase sequence of said enzyme donor.
9. A method according to claim 7 , wherein said enzyme donor is joined to a protease recognition sequence.
10. A method according to claim 8 , wherein said enzyme donor is joined to an enzyme.
11. A method according to claim 8 , wherein said enzyme donor is joined to a polypeptide in the pathway regulating transcription.
12. A kit comprising a cell according to claim 1 .