Functionalized nanoparticles for enhanced affinity precipitation of proteins
AssigneeUniversity of Delaware
InventorsWilfred Chen, Andrew Swartz
Life of this patent
Estimating the term…
Abstract
The present invention provides a nanoparticle capable of binding specifically to a target protein in a solution and precipitating with the target protein out of the solution upon addition of the target protein to the solution. The precipitation may be reversed release the target protein from the nanoparticle, which may be reused for precipitating the target protein. Also provided are a method for purifying a target protein by affinity precipitation using the nanoparticle without chromatography and a method for preparing the nanoparticle.
Claims67
A nanoparticle comprising a fusion protein and a scaffolding domain, wherein the fusion protein is covalently bound to the scaffolding domain, wherein the fusion protein comprises an affinity domain specific for a target protein and a stimuli responsive precipitation domain, wherein the scaffolding domain comprises self-assembled proteins and has a diameter of at least 10 nm, wherein the nanoparticle is soluble in a first solution in the absence of the target protein, wherein the nanoparticle is capable of binding specifically to the target protein in the first solution and precipitating with the target protein out of the first solution in response to a first stimulus, and wherein the first stimulus comprises addition of the target protein to the first solution.
A composition comprising a nanoparticle in a first solution, wherein the nanoparticle comprises a fusion protein and a scaffolding domain, wherein the fusion protein is covalently bound to the scaffolding domain, wherein the fusion protein comprises an affinity domain specific for a target protein and a stimuli responsive precipitation domain, wherein the scaffolding domain comprises self-assembled proteins and has a diameter of at least 10 nm, wherein the nanoparticle is soluble in the first solution in the absence of the target protein, wherein the nanoparticle is capable of binding specifically to the target protein in the first solution and precipitating with the target protein out of the first solution in response to a first stimulus, and wherein the first stimulus comprises addition of the target protein to the first solution.
A complex comprising a nanoparticle and a target protein, wherein the nanoparticle is bound specifically to the target protein, wherein the nanoparticle comprises a fusion protein and a scaffolding domain, wherein the fusion protein is covalently bound to the scaffolding domain, wherein the fusion protein comprises an affinity domain specific for the target protein and a stimuli responsive precipitation domain, wherein the scaffolding domain comprises self-assembled proteins and has a diameter of at least 10 nm, wherein the complex is capable of precipitating out of a first solution.
A composition comprising a complex in a first solution, wherein the complex comprises a nanoparticle and a target protein, wherein the nanoparticle is bound specifically to the target protein, wherein the nanoparticle comprises a fusion protein and a scaffolding domain, wherein the fusion protein is covalently bound to the scaffolding domain, wherein the fusion protein comprises an affinity domain specific for the target protein and a stimuli responsive precipitation domain, wherein the scaffolding domain comprises self-assembled proteins and has a diameter of at least 10 nm, wherein the complex is capable of precipitating out of the first solution.
A method for purifying a target protein produced by host cells without chromatography, comprising
- (a) separating the target protein from the host cells;
- (b) exposing the separated target protein to a nanoparticle in a first solution to form a complex of the target protein and the nanoparticle, wherein the nanoparticle comprises a fusion protein and a scaffolding domain, wherein the fusion protein is covalently bound to the scaffolding domain, wherein the fusion protein comprises an affinity domain specific for the target protein and a stimuli responsive precipitation domain, wherein the nanoparticle is bound specifically to the target protein, wherein the scaffolding domain comprises self-assembled proteins and has a diameter of at least 10 nm, whereby the complex precipitates out of the first solution;
- (c) adding the precipitated complex to a second solution;
- and
- (d) applying a stimulus to solubilize the precipitated complex in the second solution and release the target protein from the nanoparticle in the second solution, whereby the target protein is purified from the host cells.
A method for preparing a nanoparticle, comprising
- (a) making a fusion protein comprising an affinity domain specific for a target protein and a stimuli responsive precipitation domain;
- and
- (b) conjugating the fusion protein covalently to a scaffolding domain to generate a nanoparticle, wherein the scaffolding domain comprises self-assembled proteins and has a diameter of at least 10 nm, wherein the fusion protein is covalently bound to the scaffolding domain, and wherein the nanoparticle is soluble in a first solution in the absence of the target protein, wherein the nanoparticle is capable of binding specifically to the target protein in the first solution and precipitating with the target protein out of the first solution in response to a first stimulus, and wherein the first stimulus comprises addition of the target protein to the first solution.
Dependencies are inferred from the claim text.