Rapid and simple purification of elastin-like polypeptides directly from whole cells and cell lysates by organic solvent extraction
AssigneePudue Research Foundation
InventorsDavid Harley Thompson, Craig Sweet, Aayush Aayush, Ross VerHeul
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Abstract
Disclosed herein is an optimized Elastin-like peptide purification scheme from whole cell lysate that is broadly applicable to neutral, acidic or basic ELP polypeptide. The method involves the use of one or more organic solvent to extract the ELP polypeptide from whole lysate followed by optional back extraction, precipitation, hot spins, and/or dialysis to purify the target ELP polypeptide.
Claims10
A method to purify Elastin like polypeptides (ELPs) from a total cell lysate, comprising:
- a. Preparing a clarified cell lysate, wherein said cell lysate comprises at least one ELP with or without fusing to other proteins;
- b. Preparing at least one organic extractant using an organic solvent, wherein the organic solvent is selected from the group consisting of IPA, nBuOH, EtOAc, Ace, ACN, EtOH and MeOH, or a combination thereof;
- c. Adding said cell lysate to the organic extractant to form a mixture;
- d. Vortexing the mixture and subjecting the mixture to centrifugation to separate out an organic phase;
- and
- e. Selectively recovering the organic phase, which comprises said at least one ELP with or without fusing to other proteins.
A method to purify Elastin like polypeptides (ELPs) from whole cells that express at least one ELP with or without fusing to other proteins, comprising:
- a. Preparing whole cell pellets comprising at least one ELP with or without fusing to other proteins;
- b. Mixing an organic solvent with said whole cell pellets to form a first mixture, wherein the organic solvent is selected from the group consisting of IPA, nBuOH, EtOAc, Ace, ACN, EtOH and MeOH, or a combination thereof;
- c. Vortexing the first mixture and subjecting the first mixture to centrifugation until a first organic phase is formed;
- d. Selectively collecting the first organic phase;
- e. Adding anti-solvent and water to the first organic phase to form a second mixture;
- f. Subjecting the second mixture to centrifugation until an aqueous phase and a second organic phase are formed;
- and
- g. Removing the second organic phase to recover the purified (back-extracted) ELP in the aqueous phase.
A method to purify Elastin like polypeptides (ELPs) from whole cells that express at least one ELP with or without fusing to other proteins, comprising:
- a. Preparing whole cell pellets comprising at least one ELP with or without fusing to other proteins;
- b. Mixing an organic solvent with said whole cell pellets to form a first mixture, wherein the organic solvent is selected from the group consisting of IPA, nBuOH, EtOAc, Ace, ACN, EtOH and MeOH, or a combination thereof;
- c. Vortexing the first mixture and subjecting the first mixture to centrifugation until a first organic phase is formed;
- d. Selectively collecting the first organic phase;
- e. Adding acetonitrile to the collected organic phase to form a second mixture;
- f. Subjecting the second mixture to centrifugation until a pellet and a second organic phase are formed;
- g. Removing the second organic phase to recover the pelleted (precipitated) ELP and placing it back in an aqueous phase solution;
- and
- h. Subjecting the aqueous phase solution to centrifugation to remove contaminants and recovering ELP in an aqueous phase by collecting a supernatant.
Dependencies are inferred from the claim text.