Patent ID: 9169312
Filing Date: 2015-10-27
Classification: C07K,C12N,C12Q,G01N

Abstract:
1. A cell-based method for monitoring the activity of an HIV-1 protease comprising: (1) (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises: (i) an amino acid motif or subsequence susceptible to cleavage by the HIV-1 protease under physiologic (cell culture) conditions comprising SEQ ID NO:3 or SEQ ID NO:4; (ii) a transmembrane domain; (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and (iv) a detectable moiety, wherein the amino acid motif or subsequence susceptible to cleavage by the HIV-1 protease is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the HIV-1 protease the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety; (b) providing a nucleic acid encoding the HIV-1 protease operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous form of the HIV-1 protease; (c) inserting (transfecting) the nucleic acid of (a) and (b) into the cell if the cell does not already express a heterologous or endogenous form of the HIV-1 protease; (d) co-expressing the nucleic acid of (a) and (b) in the cell, or expressing the nucleic acid in the cell if the cell already expresses a heterologous or endogenous form of the HIV-1 protease; and (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell, wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the HIV-1 protease is not enzymatically active, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the enzyme is enzymatically active.