Patent ID: 9134317
Filing Date: 2015-09-15
Classification: C07K,C40B,G01N

Abstract:
1. A high throughput method for generating and screening of probes comprising the steps of: (a) providing a reference intracellular lipid binding protein (iLBP) comprising a single cysteine residue selected from the group consisting of an iLBP having the sequence set forth in SEQ ID No: 5, L10P7A4-L30C, L11P7B3-V26C, L13EP16E11, L18P5G12-K27C, L19CP10C7, L50BP4E2, L50BP9D5, L61P8B12, L68P3H10, L71AP22B3, L76P9E4, L83P5G8, and L85P1C2, wherein the reference iLBP has a binding specificity for a set of unbound analytes of interest; (b) generating polynucleotide mutants encoding a iLBP library comprising an assortment of iLBP muteins of the reference iLBP from a polynucleotide template encoding the reference iLBP; (c) expressing the iLBP muteins comprising a single cysteine residue encoded by the library; (d) incubating the expressed iLBP muteins with the solid matrix; (e) purifying the iLBP muteins by binding to the solid matrix; (f) labeling the matrix-bound iLBP muteins with a single fluorophore to produce probes at a pH of less than 8 whereby the fluorophore reacts with the cysteine side-chain and responds to analyte binding by a change in fluorescent ratio at two wavelengths, wherein the fluorophore is covalently attached to the cysteine residue only; (g) retrieving the probes with altered specificity to a set of unbound analytes of interest relative to the reference iLBP from the solid matrix; (h) screening each of the retrieved probes in a fluorometer in the presence and absence of a set of unbound analytes of interest each with defined unbound concentrations to determine binding of each probe to the set of unbound analytes of interest; (i) screening each of the retrieved probes in a fluorometer in the presence and absence of a set of unbound analytes of interest to determine binding of each probe to the set of unbound analytes of interest, wherein the response of the probes differs from the response of the reference labeled iLBP; and (j) observing a change in a ratio (R) of a fluorescence index of the probes measured at two different wavelengths in the presence and absence of the unbound analytes.