Methods and compositions for identifying a polynucleotide encoding a protease
AssigneeBioFocus Discovery Limited
InventorsMark P. Chadwick, Stephen J. Russell, Christian Buchholz
Life of this patent
Estimating the term…
Abstract
Methods are disclosed whereby protease activity is directly linked to delivery of a transferable label to a target cell which expresses a protease, via fusion of viral display packages comprising the transferable label with the target cell. The methods can be used, inter alia, to identify proteases, including previously undiscovered proteases or variants of known proteases which may have altered substrate specificity.
Claims51
A method of identifying a candidate polynucleotide molecule encoding a cell surface protease or a secreted protease, comprising the steps of:
- (a) contacting viral display packages with target cells, wherein:
- (1) the viral display packages comprise a transferable label and display a chimeric envelope protein comprising (i) a substantially intact viral envelope protein which enhances fusion between a viral display package and a target cell membrane, (ii) an inhibitory protein which prevents fusion between the viral display package and the target cell membrane, and (iii) a protease recognition site located between the substantially intact viral envelope protein and the inhibitory protein;
- and
- (2) the target cells comprise expressible candidate polynucleotide molecules, wherein the expressible candidate polynucleotide molecules may or may not encode a protease;
- and
- (b) detecting a target cell which comprises the transferable label, wherein infection of a target cell by a viral display package occurs only if a protease produced in the target cell removes the inhibitory protein from the chimeric envelope protein.
A method of identifying a candidate polynucleotide molecule encoding a cell surface protease or a secreted protease, comprising the steps of:
- (a) contacting viral display packages with target cells, wherein:
- (1) the viral display packages comprise a transferable label and display recombinant envelope proteins in which a protease recognition site has been substituted for a furin cleavage site located between a large glycoprotein subunit of the envelope protein and a transmembrane component of the envelope protein;
- and
- (2) the target cells comprise expressible candidate polynucleotide molecules, wherein the expressible candidate polynucleotide molecules may or may not encode a protease;
- and
- (b) detecting a target cell which comprises the transferable label, wherein infection of a target cell by a viral display package occurs only if a protease produced in the target cell activates the envelope protein by cleaving it at the protease recognition site between the large glycoprotein subunit and the transmembrane component.
Dependencies are inferred from the claim text.