Pyolysin methods and compositions
Methods for improved cultivation media and culture conditions for Trueperella pyogenes are disclosed herein. Also disclosed are improved methods for the isolation and purification of pyolysin from Trueperella pyogenes.
1. A method for increasing the yield of pyolysin produced by Trueperella pyogenes , wherein the method comprises: A) culturing T. pyogenes at 21 to 36 degrees C. in a basal medium that contains glucose, and, optionally, an additional concentrated carbon source selected from the group consisting of glucose, galactose, sucrose, maltose, oligosaccharides, glycerol, lactose, dextran, dextrin, mono methyl succinate, and N-acetal glucosamine; B) adding a calcium chelating agent and lactose to the culture medium prior to the exhaustion of glucose in the medium; C) harvesting T. pyogenes , and D) isolating pyolysin.
2. The method of claim 1 , wherein the additional concentrated carbon source is lactose.
3. The method of claim 1 , wherein the chelating agent is ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), or a combination of the two.
4. The method of claim 3 , wherein the chelating agent is EGTA.
5. The method of claim 1 , wherein T. pyogenes replicates to a bacterial cell density higher than an optical density (O.D.) of 5 at 600 nm.
6. The method of claim 1 , wherein the medium is maintained at a temperature between 21-36° C.
7. The method of claim 6 , wherein the medium is maintained at a temperature between 28-32° C.
8. The method of claim 1 , further comprising the use of the basal medium, wherein the Ph of the medium is between 6.0 and 8.0.
9. The method of claim 1 , further comprising the use of the basal medium, wherein the medium comprises hemin as an iron source.
10. The method of claim 1 , further comprising the use of the basal medium comprising Polysorbate 80 (Tween 80).
11. The method of claim 1 , further comprising the use of the basal medium comprising a vitamin solution.
12. The method of claim 11 , wherein the vitamin solution comprises one or more of the following: Vitamin B12; myo-inositol; uracil nucleobase; nicotinic add; calcium pantothenate; pyridoxal-HCl; pyridoxamine-2HCl; riboflavin; thiamine-HCl; p-aminobenzoic add; biotin; folic add; niacinamide; and □-NAD.
13. The method of claim 11 , wherein the vitamin solution comprises pyridoxal-HCl.
14. The method of claim 1 , further comprising allowing the culture Ph to decrease from the initial basal medium starting Ph to a level between 5.50 and 6.50, and then controlling the Ph between 5.50 and 6.50 by automatic addition of a basic titrant.
15. The method of claim 1 , further comprising separating T. pyogenes proteases from the pyolysin.
16. The method of claim 15 , wherein the separation of T. pyogenes proteases from pyolysin is achieved by a chromatography step.