Electronic protein fractionation

9,321,012

13/803564

March 14, 2013

Apparatuses and methods for purifying proteins and other target molecules based on pI are provided.

Bio-Rad Laboratories, Inc. (Hercules, CA, US); Technion Research & Development Foundation Ltd. (Technion, Haifa, IL)

Bio-Rad Laboratories, Inc. (Hercules, CA, US)

1. An apparatus, comprising a chamber divided into a first sub-chamber and a second sub-chamber by a dividing membrane, wherein the dividing membrane blocks or substantially blocks flow of fluid between the first and second sub-chamber; and wherein the first sub-chamber is in electrical and fluid communication with a first ion injector extractor comprising a first electrode; the second sub-chamber is in electrical and fluid communication with a second ion injector extractor comprising a second electrode; and the second sub-chamber comprises an outlet, wherein the ion injector/extractors each comprise: a. a compartment in fluid communication with a sub-chamber and divided from the sub-chamber by an anion selective membrane, wherein the anion selective membrane is permeable to small anions but not larger molecules; and/or b. a compartment in fluid communication with the sub-chamber and divided from the sub-chamber by a bipolar membrane, wherein the bipolar membrane is permeable to small ions but not larger molecules; and/or c. compartment in fluid communication with a sub-chamber and divided from the sub-chamber by a cation selective membrane, wherein the cation selective membrane is permeable to small cations but not larger molecules.

2. The apparatus of claim 1 , wherein the second sub-chamber is smaller than the first sub-chamber.

3. The apparatus of claim 1 , wherein the first electrode is an anode and the second electrode is a cathode.

4. The apparatus of claim 1 , wherein the first electrode is a cathode and the second electrode is an anode.

5. The apparatus of claim 1 , wherein the injector/extractors are directly linked to the respective sub-chambers.

6. The apparatus of claim 1 , wherein the injector/extractors are linked to the respective sub-chambers via tubing or a channel.

7. The apparatus of claim 1 , wherein the second sub-chamber further comprises one or more inlet.

8. The apparatus of claim 1 , wherein the dividing membrane comprises a layer of cross-linked polymer, thereby inhibiting fluid diffusion.

9. The apparatus of claim 1 , wherein the dividing membrane contains pores that allow passage of peptides smaller than a molecular cut-off but substantially block passage of peptides larger than the molecular cut-off.

10. The apparatus of claim 9 , wherein the molecular cut-off is about 20 kDa.

11. A method of purifying a target protein(s) or peptide(s) from a sample, the method comprising providing the apparatus of claim 1 ; loading the sample into the first sub-chamber, or the first and second sub-chambers, wherein the first and second sub-chambers contain fluid following the loading, controlling the injector/extractors to adjust the pH of the fluid in the chamber to a pH such that some components of the sample are charged due to the pH adjustment and some components are not charged; applying a voltage between the first and second electrode, thereby moving at least some charged components into the second sub-chamber; and removing the fluid in the second chamber including the charged components via the outlet in the second sub-chamber, thereby separating the target protein(s) or peptide(s) in the sample from at least some other components of the sample.

12. The method of claim 11 , wherein one or more target protein or peptide is a charged component moved to the second sub-chamber, and the target protein or peptide is collected after the removing.

13. The method of claim 11 , wherein the charged components moved to the second sub-chamber are contaminants and discarding the contaminants.

14. The method of claim 11 , wherein the sample is loaded into the first sub-chamber only.

15. The method of claim 11 , wherein the sample is loaded into the first and second sub-chambers.

16. The method of claim 11 , wherein the first electrode is a cathode (negative charge) and the second electrode is an anode (positive charge), and the controlling comprises adjusting the pH of the fluid below the pI of the target protein(s) or peptide(s) such that the target protein(s) or peptide(s) have an overall positive charge and at least some other components of the sample are negatively-charged; and the applying results in movement of the negatively-charged components to the second sub-chamber and the target protein(s) or peptide(s) remain in the first sub-chamber; and removing fluid comprising the moved components from the second sub-chamber and optionally replacing the removed fluid with new fluid in the second sub-chamber; and subsequently controlling the injector/extractors to adjust the pH of the fluid in the chamber to a pH above the pI of the target protein(s) or peptide(s) such that the target protein(s) or peptide(s) have an overall negative charge; applying a voltage between the first and second electrode, thereby moving the negatively-charged target protein(s) or peptide(s) into the second sub-chamber; and removing and collecting the fluid in the second chamber, including the target protein(s) or peptide(s), via the outlet in the second sub-chamber, thereby separating the target protein(s) or peptide(s) in the sample from at least some other components of the sample.

17. The method of claim 11 , wherein the first electrode is an anode (positive charge) and the second electrode is a cathode (negative charge), and the controlling comprises adjusting the pH of the fluid above the pI of the target protein(s) or peptide(s) such that the target protein(s) or peptide(s) have an overall negative charge and at least some other components of the sample are positively-charged; and the applying results in movement of the positively-charged components to the second sub-chamber and the target protein(s) or peptide(s) remain in the first sub-chamber; and removing fluid comprising the moved components from the second sub-chamber and optionally replacing the removed fluid with new fluid in the second sub-chamber; and subsequently controlling the injector/extractors to adjust the pH of the fluid in the chamber to a pH below the pI of the target protein(s) or peptide(s) such that the target protein(s) or peptide(s) have an overall positive charge; applying a voltage between the first and second electrode, thereby moving the positively-charged target protein(s) or peptide(s) into the second sub-chamber; and removing and collecting the fluid in the second chamber, including the target protein(s) or peptide(s), via the outlet in the second sub-chamber, thereby separating the target protein(s) or peptide(s) in the sample from at least some other components of the sample.

18. The method of claim 11 , wherein the sample comprises proteins and the dividing membrane contains pores that allow passage of peptides smaller than a molecular cut-off but that substantially block passage of peptides larger than the cut-off, and the loading comprises loading the sample into the first sub-chamber, optionally, the controlling comprises controlling the injector/extractors to adjust the pH of the fluid in the first sub-chamber; adding a first protease to the first-sub chamber under conditions to allow the first protease to generate peptides from the proteins; applying a voltage between the first and second electrode, thereby moving at least some charged peptides having a size below the molecular cut-off into the second sub-chamber.

19. The method of claim 18 , wherein the molecular cut-off is about 10, 15, 20, 25, or 30 kDa.

20. The method of claim 18 , further comprising, adding a second protease to the first sub-chamber under conditions to allow the second protease to generate peptides from the proteins; applying a voltage between the first and second electrode, thereby moving at least some charged peptides having a size below the molecular cut-off into the second sub-chamber.

21. The method of claim 20 , comprising, before or after adding the second protease, adjusting the pH of the fluid in the first sub-chamber to a pH optimized for the second protease.

22. The method of claim 18 , wherein before the adding of the first protease, the method comprises applying a voltage between the first and second electrode, thereby moving at least some charged peptides, if present from the sample, into the second sub-chamber.

23. The method of claim 18 , further comprising collecting the peptides in the second sub-chamber.

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