PEGBOARD PROTEIN PURIFICATION PLATE FOR HIGH THROUGHPUT PROTEIN CHARACTERIZATION

Abstract

Disclosed herein are methods and compositions for a new way to separate biologics using coated pegboardsplastic plates affixed with an array of 96-pegs which align with the wells of a traditional 96-well plate used in bench top experiments. The ends of the pegs are conjugated to various compounds which are able to bind tags on engineered proteins (ex. Ni-NTA would bind a His-Tag on an engineered protein). These pegs extend into plate wells to specifically bind the tagged protein from the cell lysate which contains hundreds of other proteins. The pegboard, now laden with the protein of interest (POI), may then be dipped into sequential buffers to wash away potential non-specifically bound proteins to purify the POI.

Claims

1. A system for using plates affixed with an array of pegs which interact with the wells of a well plate; wherein the pegs are conjugated to compounds which bind tags on an engineered biological moiety; and wherein the pegs extend into the well plate to bind the tags on the engineered biological moiety.

2. The system of claim 1 wherein the engineered biological moiety is part of a cell lysate.

3. The system of claim 1 wherein the engineered biological moiety is a DNA molecule.

4. The system of claim 1 wherein the engineered biological moiety is a RNA molecule.

5. The system of claim 1 wherein the engineered biological moiety is a protein.

6. The system of claim 1 wherein the tags on the engineered biological moiety comprise FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin system tags.

7. The system of claim 3 wherein the tags on the engineered DNA molecule comprise FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin system tags.

8. The system of claim 4 wherein the tags on the engineered RNA molecule comprise FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin system tags.

9. The system of claim 5 wherein the tags on the engineered protein comprise FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin system tags.

10. A method for purifying an engineered biological moiety using plates affixed with an array of pegs which interact with the wells of a well plate; wherein the pegs are conjugated to compounds which bind tags on an engineered biological moiety; and wherein the pegs extend into the well plate to bind the tags on the engineered biological moiety; and wherein the method comprises the step of incubating the pegs in a cell-extract solution within the well plate; and wherein the cell-extract solution comprises an engineered biological moiety.

11. The method of claim 10 wherein the engineered biological moiety is part of a cell lysate.

12. The method of claim 10 wherein the engineered biological moiety is a DNA molecule.

13. The method of claim 10 wherein the engineered biological moiety is a RNA molecule.

14. The method of claim 10 wherein the engineered biological moiety is a protein.

15. The method of claim 10 wherein the tags on the engineered biological moiety comprise FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin system tags.

16. The system of claim 12 wherein the tags on the engineered DNA molecule comprise FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin system tags.

17. The system of claim 13 wherein the tags on the engineered RNA molecule comprise FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin system tags.

18. The system of claim 14 wherein the tags on the engineered protein comprise FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin system tags.

19. The method of claim 10 further comprising washing the incubated pegs to remove non-specific binding of non-engineered biological moieties.

20. The method of claim 10 further comprising eluting the bound biological moieties from the pegs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 depicts a system of using a purification pegboard for high throughput purification of enzymes. The system essentially acts like a stamp, has dimensions of 96-well (universally compatible with current labware), exhibits one peg per well, and is surface coated with a compound to bind proteins. Various peg geometries are contemplated which fit flat, or PCR 96-well plates which can also tune binding capacity (i.e., peg surface area).

[0010] FIG. 2 depicts a flow chart of an embodiment of a method disclosed herein which is useful for the high throughput purification of proteins using compositions and systems disclosed herein. In an embodiment, 96 samples of purified protein are produced in less than about 20 min which is easy to perform in parallel and exhibits decreased error and is capable of purifying low volumes.

[0011] FIGS. 3A, and 3B depict plate geometries contemplated in embodiments disclosed herein.

[0012] FIGS. 4A, 4B, 4C, 4D and 4E depict contemplated peg designs to suit corresponding plate geometries. FIG. 4A depicts a rounded-cone peg which pushes fluid up around edges to increase surface/binding area. FIG. 4B depicts a hollow cylinder peg with high surface area for binding plate-readers and can be used with peg still in the plate, for example. FIG. 4C depicts a solid cylinder peg which maximizes contact area with small sample volumes and exhibits an easily tunable surface area to tailor binding capacity. FIG. 4D depicts a cone shape with no-base peg which maximizes contact area with small sample volumes and exhibits an easily tunable surface area to tailor binding capacity. FIG. 4E depicts a cone shape with beveled edges peg which maximizes contact area with small sample volumes and exhibits an easily tunable surface area to tailor binding capacity.

[0013] FIGS. 5A, 5B depict (FIG. 5A) AFU over time, BDH bound to Pegboard; and (FIG. 5B) A420 over time POD bound to Pegboard. As depicted in FIGS. 5A and 5B, the Pegboard binds protein and allows facile activity assays in high throughput for non-functionalized (bare plastic)BDH which binds protein from buffer; allows for functionalized (FSL-biotin coated plastic)POD which binds protein from buffer; concentration dependent activity; and works for different proteins and reactions.

DETAILED DESCRIPTION

[0014] In an embodiment, disclosed herein are methods and compositions of matter comprising DNA, RNA and protein purification. In an embodiment, the methods disclosed herein represent a new way to separate biologics using coated pegboards on plastic plates affixed with an array of 96-pegs which align with the wells of a traditional 96-well plate used in bench top experiments. The ends of the pegs are conjugated to various compounds which are able to bind tags on engineered proteins (ex. Ni-NTA would bind a His-Tag on an engineered protein). In additional embodiments, the ends of the pegs are conjugated to various compounds which are able to bind tags on engineered biological moieties comprising RNA molecules, DNA molecules and proteins of interest. These pegs extend into the bottom of the plate wells to specifically bind the tagged protein from the cell lysate which contains hundreds of other proteins. The pegboard, now laden with the protein of interest (POI), may then be dipped into sequential buffers to wash away potential non-specifically bound proteins to purify the POI. Finally, the pegboard can be dipped into a plate containing reaction buffer where the POI activity can be directly measured, or otherwise may be eluted off of the peg to generate stocks of purified protein.

[0015] Benefits over existing techniques include less plastic waste by using fewer pipette tips and reusing the binding plates; saving time by handling 96 samples at a time resulting in a less than 20 minute purification system that exhibits the potential for facile integration into a robotics platform; tunable binding capacity that can be used to normalize protein levels for assays; and can be expanded to use with a broad range of affinity tags including, but not limited to, FLAG, HPC, GST, His-Tag, Strep II, and avidin-biotin systems.

[0016] In an embodiment, the system and device of coated pegboards can be made of a variety of materials or methods to tailor surface area to the application. 3D printing leaves a porous surface texture vs smooth from injection molding.

[0017] In an embodiment, a bound protein on the pegboard can be reused for multiple rounds of assay (dictated by the lifetime of the enzyme).

[0018] In an embodiment, uncoated plastic may be used to make the Pegboard. This could allow for an activity assay of adsorbed or bound protein that has been pre-purified. An additional benefit is that this embodiment could be useful for testing different reaction conditions.

[0019] In an embodiment, this system and device of coated pegboards affixed to plates is able to assist in high-throughput protein purification. With the pegboard, 96-enzyme mutants can be manipulated at once, and purification time is much quicker compared to traditional protein-purification methods, making the whole process of testing enzyme mutants much more rapid. In an embodiment, the system can be used to tailor the binding capacity of each peg for a POI allowing for tighter control of experimental conditions, and normalizing protein levels when assaying activity.

[0020] In an embodiment, the devices disclosed herein are able to assist in high-throughput protein purification. With the pegboard, 96-enzyme mutants can be manipulated at once, and purification time is much quicker compared to traditional protein-purification methods, making the whole process of testing enzyme mutants much more rapid. In an embodiment, this invention is uniquely able to tailor the binding capacity of each peg for the POI, allowing for tighter control of experimental conditions, and normalizing protein levels when assaying activity.

[0021] In an embodiment, the methods, systems and compositions of matter disclosed herein require only the plastic plate coated in a binding-compound for the POI. In an embodiment, the methods, systems and compositions of matter disclosed herein would be able to be automated for robotics platforms. In an embodiment, the plate is reused with thorough washing, and re-charging of the bind chemical to reduce plastic waste.

[0022] Disclosed herein are novel plate designs and geometries of pegs useful for high throughput purification of biologics such as proteins.

[0023] The foregoing disclosure has been set forth merely to illustrate the invention and is not intended to be limiting.