Protein arrays and methods of using and making the same
Inventors
Jacobson, Joseph • Hudson, Michael E. • Chu, Larry Li-Yang
Assignees
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Publication Number
US-10457935-B2
Publication Date
2019-10-29
Expiration Date
Abstract
Methods and devices are provided for preparing a protein array having a plurality of proteins. In one embodiment, the method includes providing a plurality of nucleic acids each having a predefined sequence and expressing in vitro a plurality of proteins from the plurality of nucleic acids. In another embodiment, protein arrays having a solid surface and a microvolume are also provided. The solid surface can have a plurality of anchor oligonucleotides capable of hybridizing with a plurality of nucleic acids. The microvolume can cover each of the plurality of anchor oligonucleotides and can be configured to produce a polypeptide from each of the plurality of nucleic acids.
Core Innovation
The invention relates to preparing a protein array having a plurality of proteins by using nucleic acids with a predefined sequence. The nucleic acids encode proteins of interest and are immobilized on discrete features of a surface, so that each protein corresponds to a discrete feature.
The method expresses in vitro a plurality of proteins of interest from the immobilized nucleic acids. The proteins are each present in solution at the discrete features on the surface, enabling a protein array in which protein identity is spatially associated with the corresponding immobilized nucleic acid feature.
Each protein of interest is expressed in a self-contained volume of 1 picoliter to 1 nanoliter. The disclosed approach supports microvolume, self-contained reaction volumes, including micro-well and microfluidic droplet formats, and includes options for measuring protein activity and screening.
Claims Coverage
The disclosed claim set includes one independent method claim and dependent claims that refine the independent claim by adding protein activity measurement, specifying nucleic acid synthesis on a solid surface, adding a regulatory genetic sequence, constraining the expression format to a micro-well plate, and constraining expression to different discrete features of the solid surface. Across these claims, the independent inventive structure comprises immobilized predefined-sequence nucleic acids on discrete surface features and in vitro expression so each protein is present in solution at its corresponding feature in a self-contained picoliter-to-nanoliter volume.
Immobilized predefined-sequence nucleic acids on discrete surface features
Providing a plurality of nucleic acids each having a predefined sequence and each encoding a protein of interest, wherein the plurality of nucleic acids are immobilized on discrete features of a surface.
In vitro expression to produce solution-phase proteins at discrete features
Expressing in vitro a plurality of proteins of interest from the plurality of nucleic acids, wherein the plurality of proteins are each present in solution at the discrete features on the surface.
Self-contained picoliter-to-nanoliter expression volumes
Wherein each of the plurality of proteins of interest is expressed in a self-contained volume having a size of 1 picoliter to 1 nanoliter.
Measuring an activity of proteins present in solution
Measuring an activity of each of the plurality of proteins present in solution.
Synthesis of nucleic acids on a solid surface
Providing that the plurality of nucleic acids is synthesized on a solid surface.
Nucleic acids comprising a regulatory genetic sequence
Providing nucleic acids where each contains a regulatory genetic sequence.
In vitro expression in a micro-well plate well
Expressing each protein of interest in vitro in a well of a micro-well plate.
In vitro expression at different discrete solid-surface features
Expressing each protein of interest in vitro at a different feature of a solid surface.
Overall claim coverage centers on creating a protein array by immobilizing predefined-sequence nucleic acids encoding proteins of interest on discrete surface features and expressing the proteins in vitro so each protein is present in solution at its corresponding feature within a self-contained 1 picoliter to 1 nanoliter volume. Dependent refinements include measuring protein activity, synthesizing nucleic acids on a solid surface, adding a regulatory genetic sequence, and constraining the expression format to micro-well plate wells and/or different discrete solid-surface features.
Stated Advantages
Enables preparation of a protein array with multiple proteins associated with discrete surface features via in vitro expression.
Uses self-contained 1 picoliter to 1 nanoliter volumes for expressing each protein of interest.
Allows measurement of protein activity for proteins present in solution.
Documented Applications
Protein array fabrication supporting measurement of protein activity.
High-throughput protein expression and assays using microvolume formats and feature-based expression.
Combinatorial multi-enzyme pathway development and enzymatic pathway screening using microarrayed gene combinations.
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