Method for automated protein purification
View Patent ↗Methods and systems for automated protein purification are disclosed. The disclosed methods and systems utilize a plurality of magnetic bead chemistries and an automated extraction system for the purification of one or more proteins within a short time frame.
1. A method for purification of a target protein fused to an affinity tag, comprising:
contacting a sample comprising the target protein with a plurality of type 1 magnetic beads capable of recognizing and binding the affinity tag under conditions sufficient for the plurality of type 1 magnetic beads to bind to the affinity tag fused to the target protein in the sample, wherein a protease cleavage sequence is between the affinity tag and the target protein;
washing the plurality of type 1 magnetic beads to remove contaminating material from the sample;
contacting the sample with a cleavage solution containing an endoprotease that cleaves the cleavage sequence to release the target protein from the plurality of type 1 magnetic beads;
contacting the sample with a plurality of type 2 magnetic beads capable of recognizing and binding to the endoprotease under conditions sufficient to bind the endoprotease in the sample, wherein the type 2 magnetic beads do not bind the affinity tag fused to the target protein; and
removing the plurality of type 1 magnetic beads and the plurality of type 2 magnetic beads, thereby purifying the target protein.
2. The method of claim 1 , wherein washing the plurality of type 1 magnetic beads further comprises:
contacting the plurality of type 1 magnetic beads that are bound to the affinity tag fused to the target protein with a magnet under conditions sufficient to bind the plurality of type 1 magnetic beads to the magnet; and
transferring the plurality of type 1 magnetic beads that are bound to the affinity tag fused to the target protein to one or more wash solutions to facilitate removal of contaminating material from the sample; and
subsequent to the washing, releasing the plurality of type 1 magnetic beads from the magnet.
3. The method of claim 1 , further comprising:
removing the plurality of type 1 magnetic beads from the sample prior to contacting the sample with the plurality of type 2 magnetic beads.
4. The method of claim 1 , wherein contacting the sample with the plurality of type 2 magnetic beads occurs without prior removal of the plurality of type 1 magnetic beads from the sample.
5. The method of claim 1 , wherein the affinity tag fused to the target protein is a first affinity tag; and
wherein the protease cleaves the target protein from the first affinity tag, thereby releasing the target protein from the plurality of type 1 magnetic beads, while the first affinity tag remains bound to the plurality of type 1 magnetic beads.
6. The method of claim 5 , wherein the plurality of type 2 magnetic beads bind to the endoprotease by way of a second affinity tag fused to the endoprotease.
7. The method of claim 6 , wherein the first affinity tag and the second affinity tag are different.
8. The method of claim 5 , wherein the target protein is comprised of two or more different proteins; and
wherein the endoprotease further cleaves the target protein into the two or more different proteins.
9. The method of claim 1 , wherein the method does not include any centrifugation steps.
10. The method of claim 1 , wherein the method does not involve use of any detergent.