Tandem fluorescent protein constructs

A method for determining whether a sample contains an enzyme comprising:

• contacting the sample with a tandem fluorescent protein construct which comprises a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a linker moiety that couples the donor and acceptor moieties and that comprises a cleavage recognition site specific for the enzyme, wherein the donor and acceptor moieties exhibit fluorescence resonance energy transfer when the donor moiety is excited;
• exciting the donor moiety;
• and
• determining the degree of fluorescence resonance energy transfer in the sample, whereby a degree of fluorescence resonance energy transfer that is lower than an expected amount indicates the presence of an enzyme.

A method of determining the amount of activity of an enzyme in a cell comprising the steps of:

• providing a cell that expresses a tandem fluorescent protein construct, the construct comprising a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a peptide linker moiety, wherein the peptide linker moiety comprises a cleavage recognition amino acid sequence specific for the enzyme, and wherein the donor and acceptor moieties exhibit fluorescence resonance energy transfer when the donor moiety is excited;
• exciting the donor moiety;
• and
• determining the degree of fluorescence resonance energy transfer in the cell, whereby the degree of fluorescence resonance energy transfer relates the amount of enzyme activity in the cell.

A method of determining the amount of activity of an enzyme in a sample from an organism comprising the steps of:

• providing a sample from an organism having a cell that expresses a tandem fluorescent protein construct, the construct comprising a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a peptide linker moiety, wherein the peptide linker moiety comprises a cleavage recognition amino acid sequence specific for the enzyme, and wherein the donor and acceptor moieties exhibit fluorescence resonance energy transfer when the donor moiety is excited;
• exciting the donor moiety;
• and
• determining the degree of fluorescence resonance energy transfer in the sample, whereby the degree of fluorescence resonance energy transfer reflects the amount of enzyme activity in the cell.

A method for determining whether a compound alters the activity of an enzyme comprising the steps of:

• contacting a sample containing a known amount of the enzyme with the compound and with a tandem fluorescent protein construct which comprises a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a linker moiety that couples the donor and acceptor moieties and that comprises a cleavage recognition site specific for the enzyme, wherein the donor and acceptor moieties exhibit fluorescence resonance energy transfer when the donor moiety is excited;
• exciting the donor moiety;
• and
• determining the amount of enzyme activity in the sample as a function of the degree of fluorescence resonance energy transfer in the sample.

A method for determining whether a compound alters the activity of an enzyme in a cell comprising the steps of:

• providing first and second cells that express a tandem fluorescent protein construct, the construct comprising a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a peptide linker moiety, wherein the peptide linker moiety comprises a cleavage recognition amino acid sequence specific for the enzyme, and wherein the donor and acceptor moieties exhibit fluorescence resonance energy transfer when the donor moiety is excited;
• contacting the first cell with an amount of the compound;
• contacting the second cell with a different amount of the compound;
• exciting the donor moiety in the first and second cell;
• determining the degree of fluorescence resonance energy transfer in the first and second cells;
• and
• comparing the degree of fluorescence resonance energy transfer in the first and second cells, whereby a difference in the degree of fluorescence resonance energy transfer indicates that the compound alters the activity of the enzyme.

A method of testing for cleavage enzyme activity comprising:

• contacting a cleavage enzyme with a tandem fluorescent protein construct which comprises a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a linker moiety that couples the donor and acceptor moieties and that comprises at least one cleavage recognition site, wherein the donor and acceptor moieties exhibit fluorescence resonance energy transfer when the donor moiety is excited;
• exciting the donor moiety;
• and
• determining the presence of fluorescence resonance energy transfer between the donor and the acceptor moieties,
• wherein a decrease in fluorescence resonance energy transfer indicates the presence of a cleavage recognition site that is cleaved by the cleavage enzyme.