Method and device for improving protein stability and solubility
View Patent ↗A method for expressing proteins as a fusion chimera with a domain of p26 or alpha crystallin type proteins to improve the protein stability and solubility when over expressed in bacteria such as E. coli is provided. Genes of interest are cloned into the multiple cloning site of the Vector System just downstream of the p26 or alpha crystallin type protein and a thrombin cleavage site. Protein expression is driven by a strong bacterial promoter (TAC). The expression is induced by the addition of 1 mM IPTG that overcomes the lac repression (lac I q ). The soluble recombinant protein is purified using a fusion tag.
1. A method for producing soluble and active recombinant protein comprising:
a) expressing an insoluble protein as a fusion protein with an alpha-crystallin type protein or a fragment thereof in bacteria;
b) purifying said fusion protein; and
c) removing said alpha-crystallin type protein or fragment thereof from said purified fusion protein,
thereby resulting in said soluble and active recombinant protein.
2. The method of claim 1 , wherein said alpha-crystallin type protein is selected from the group consisting of p26, SicA, and alpha-A-crystallin.
3. The method of claim 1 , wherein said fusion protein comprises said alpha-crystallin type protein or a fragment thereof that has an ability of improving stability and solubility of said insoluble protein, said insoluble protein, and a proteolytic cleavage site, and wherein said cleavage site is positioned between said alpha-crystallin type protein or a fragment thereof that has an ability of improving stability and solubility of said insoluble protein and said insoluble protein.
4. A method of increasing the solubility of a first protein, comprising expressing said first protein as fusion protein with a second protein consisting essentially of an alpha-crystallin type protein or a fragment thereof that has an ability of increasing the solubility of said first protein.
5. The method of claim 4 , wherein said alpha-crystallin type protein is selected from the group consisting of p26, SicA, and alpha-A-crystallin.
6. The method of claim 4 , wherein said fusion protein comprises said alpha-crystallin type protein or a fragment thereof that has an ability of increasing the solubility of said first protein, said first protein, and a proteolytic cleavage site, an wherein said cleavage site is positioned between said alpha-crystallin type protein or a fragment thereof that has an ability of increasing the solubility of said first protein and said first protein.
7. A method of increasing the stability of a first protein, said method comprising:
a) expressing said first protein as a fusion protein with a second protein consisting essentially of an alpha-crystallin type protein in bacteria;
b) purifying said fusion protein; and
c) removing said alpha-crystallin type protein or a fragment thereof that has an ability of increasing the stability of said first protein from said purified fusion protein,
thereby resulting in said first protein.
8. The method of claim 7 , wherein said alpha-crystallin type protein is alpha-A-crystallin, and said fusion protein comprises said alpha-A-crystallin, said first protein, and a proteolytic cleavage site, and wherein said cleavage site is positioned between said alpha-A-crystallin protein and said first protein.
9. A method for purifying native bovine alpha-crystallin protein, said method comprising the steps of:
a) contacting a protein fraction comprising the bovine alpha-crystallin protein with a glycine solution having a pH of approximately 2.5;
b) size filtering the fraction of step a) by chromatography;
c) neutralizing the fraction containing the bovine alpha-crystallin protein; and
d) dialyzing the fraction containing the bovine alpha-crystallin protein into a buffer comprising 50% glycerol and having a pH of approximately 8.
10. A method for protecting a protein from proteolysis during purification, said method comprising applying a sample comprising said protein to a chromatographic pre-column filter, said filter comprising bovine alpha-crystallin protein that is coupled to a chromatography resin.