Optogenetic construct for allosteric control of protein activity
Inventors
Assignees
Bd Of Trustees Of University Of Illinois • University of Illinois System
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Publication Number
US-11965002-B2
Publication Date
2024-04-23
Expiration Date
Abstract
A light-activated construct composed of two photoreceptive polypeptide domains connected to one another by a flexible linker is provided, as is a fusion protein containing the same and polynucleotides encoding said construct and fusion protein. Methods of making and using the light-activated construct and fusion protein are also provided.
Core Innovation
The document describes a light-responsive allosteric switch formed from two flexibly linked photoreceptive domains incorporated into a protein of interest. Light-induced dimerization and conformational change reversibly modulate protein activity; the description states that this construct can suppress activity in darkness and restore it upon illumination. Neurospora crassa VVD is given as an example of the photoreceptive domain.
The supplied content describes applications to non-receptor tyrosine kinases, non-receptor tyrosine phosphatases, and Cre recombinase, as methods and fusion proteins. It also reports Src-related experiments showing light-dependent, repeatable changes in phosphorylation-related signaling and cell behaviors. The background section is not included in the supplied content, so the problem being solved there is not explicitly described in patent.
Claims Coverage
The supplied relevant claims identify eight independent claims across six inventive features: four methods of regulating protein activity and four fusion-protein claims. Together, they cover light-responsive constructs for kinases, phosphatases, and Cre recombinase.
Light regulation of non-receptor tyrosine kinase activity
Claims 1 and 12 cover methods that insert a light-responsive construct into a non-receptor tyrosine kinase and use light-induced conformational modulation to regulate kinase activity. Claim 1 specifies identical photoreceptive domains that homodimerize; claim 12 specifies domains capable of dimerizing.
Non-receptor tyrosine kinase fusion protein
Claims 6 and 17 cover a fusion protein comprising a non-receptor tyrosine kinase with an inserted light-responsive construct containing two flexibly linked photoreceptive domains. Claim 6 specifies identical domains that homodimerize; claim 17 specifies domains capable of dimerizing.
Light regulation of non-receptor tyrosine phosphatase activity
Claim 19 covers a method that inserts a light-responsive construct into a non-receptor tyrosine phosphatase and uses light-induced conformational modulation to regulate phosphatase activity. The construct contains two flexibly linked photoreceptive domains capable of dimerizing.
Non-receptor tyrosine phosphatase fusion protein
Claim 24 covers a fusion protein comprising a non-receptor tyrosine phosphatase with an inserted light-responsive construct containing two flexibly linked photoreceptive domains capable of dimerizing.
Light regulation of Cre recombinase activity
Claim 27 covers a method that inserts a light-responsive construct into Cre recombinase and uses light-induced conformational modulation to regulate its activity. The construct contains identical, flexibly linked photoreceptive domains that homodimerize.
Cre recombinase fusion protein
Claim 30 covers a fusion protein comprising Cre recombinase with an inserted light-responsive construct containing identical, flexibly linked photoreceptive domains that homodimerize.
The independent claims cover light-responsive regulation methods and corresponding fusion proteins for non-receptor tyrosine kinases, non-receptor tyrosine phosphatases, and Cre recombinase. Their shared feature is a construct with linked photoreceptive domains whose dimerization and resulting conformational modulation are associated with regulating protein activity.
Stated Advantages
The claims describe reversible, light-responsive allosteric regulation of kinase, phosphatase, and Cre recombinase activity.
Documented Applications
Regulating non-receptor tyrosine kinase activity, including phosphorylation-related activity, using a light-responsive construct.
Regulating non-receptor tyrosine phosphatase activity using a light-responsive construct.
Regulating Cre recombinase activity using a light-responsive construct.
The description reports Src-related experiments involving light-dependent changes in phosphorylation-related signaling, cell area, protrusive activity, and polarization.
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