Reuse of intein-bound resins for protein purification
The present disclosure relates to methods of protein purification by attaching an intein-C fragment to a target protein, passing a sample containing the intein-C tagged protein over a chromatographic resin carrying an intein-N fragment so as to create an intein-N intein-C complex, releasing the target protein from the intein-C fragment, and regenerating the column under conditions that disrupt the intein-N intein-C complex while preserving column functionality for multiple reuses.
1 . A method for purifying a target molecule in a sample, the method comprising the steps of
(a) providing a sample containing a fusion protein comprising an intein-C polypeptide joined to a target molecule by a peptide bond (intein-C tagged target molecule);
(b) contacting the sample with a chromatography resin comprising a covalently-linked N-terminal intein polypeptide, under conditions in which the intein-C polypeptide in the fusion protein binds to the intein-N polypeptide in the resin to form an intein complex;
(c) optionally washing the resin containing the intein complex to remove unbound contaminants;
(d) exposing the intein complex to conditions sufficient to release the target molecule from the intein-C polypeptide;
(e) regenerating the chromatographic resin by contacting the resin with a composition having a pH in aqueous solution of about 1 to about 4;
so as to disrupt the intein-N intein-C complex and release the intein-C polypeptide from the chromatography resin; and
(f) optionally, performing at least one additional purification cycles by repeating steps (a) to (e) at least once,
wherein the regenerated chromatography resin obtained from step (e) or optional step (f) retains at least about 60%, preferably at least about 70% and more preferably at least about 80% of its C-terminal intein binding capacity after each purification cycle.
2 . The method according to claim 1 , wherein in step (e) the aqueous solution has a pH of about 1 to about 2.
3 . The method according to claim 1 , wherein step (e) further comprises regenerating the chromatographic resin by contacting the resin with at least one detergent.
4 . The method according to claim 1 , wherein step (e) further comprises regenerating the chromatographic resin by contacting the resin with a composition comprising at least one kosmotropic agent.
5 . The method according to claim 1 , wherein step (e) further comprises regenerating the chromatographic resin by contacting the resin with a composition comprising at least one chaotropic agent.
6 . The method according claim 1 , further comprising the step of performing at least one purification cycle wherein the resin is contacted with a basic composition having a pH in aqueous solution of about 9 or higher.
7 . The method according to claim 1 , wherein the intein-C tagged target molecule is prepared by attaching an intein-C polypeptide to a target molecule to obtain a fusion protein, and expressing the fusion protein in an expression system.
8 . The method according to claim 1 , wherein step (b) comprises contacting the chromatography resin with a cell culture supernatant comprising the intein-C tagged target molecule; or wherein step (b) comprises loading the intein-C tagged target molecule in a saline buffer having a pH of about 8 to about 10.
9 . The method according to claim 1 , wherein step (c) is performed, and comprises washing the chromatography resin with a washing buffer prior to releasing the target molecule from the intein-C polypeptide.
10 . The method according to claim 1 , wherein step (d) comprises contacting the resin with a cleavage buffer having a pH of about 6 to about 8, so as to release the target molecule from the intein-C polypeptide.
11 . The method according to claim 1 , wherein the composition having a pH in aqueous solution of about 1 to about 4 comprises an acidic buffer comprising an acid selected from the group consisting of phosphoric acid, glycine, hydrochloric acid, hydrobromic acid, citric acid, acetic acid, formic acid, lactic acid, carbonic acid, succinic acid, nitric acid, malic acid, oxalic acid, salicylic acid, formic acid, and any combinations thereof.
12 . The method according to claim 3 ,
wherein the detergent is selected from the group consisting of anionic detergents, cationic detergents, non-ionic detergents and zwitter ionic detergents.
13 . The method according to claim 1 , further comprising the step of isolating the target molecule obtained from step (d).
14 . The method according to claim 1 , wherein the chromatography resin is polymer based or includes a polymer.
15 . The method of claim 14 , wherein the chromatography resin is formulated on a solid support, wherein the solid support is a bead or a membrane.
16 . The method according to claim 1 , wherein the target molecule is a protein.
17 . The method according to claim 1 , wherein the intein-N polypeptide is attached to the chromatography resin through a functional group selected from the group consisting of hydroxyl, thiol, epoxide, amino, carbonyl epoxide and carboxylic acid.
18 . The method according to claim 12 , wherein the detergent is selected from polysorbates, polyethylene glycols, glycosides, poloxamers, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate, 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, alkylbenzenesulfonates, quaternary ammonium salts, and bile acids.
19 . The method according to claim 4 , wherein the kosmotropic agent is an ionic kosmotropic agent or a nonionic kosmotropic agent selected from the group consisting of carbohydrates, amino acids and alcohols.
20 . The method according to claim 5 , wherein the chaotropic agent is selected from the group consisting of guanidinium chloride, arginine, n-butanol, ethanol, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, and any combination thereof.