Scaleable manufacturing process for cysteine endoprotease B, isoform 2
Methods are provided for the production of gram to kilogram quantities of pro-EP-B2 (proenzyme form of EP-B2) in a lyophilized form. The methods include scalable fermentation, refolding and purification processes, which processes may be combined with lyophilization to yield a stable product.
1. A method for producing a self-activating Hordeum vulgare cysteine endoprotease B, isoform 2 proenzyme (proEP-B2), the method comprising:
producing proEP-B2 in a fermentation reaction with a microbial host;
extracting inclusion bodies containing proEP-B2 from said microbial host,
solubilizing the inclusion bodies in 5 M or greater urea to produce solubilized, denatured proEP-B2;
purifying said solubilized, denatured, proEP-B2; and
refolding said purified, solubilized, denatured pro-EP-B2 by rapid dilution into buffer comprising not more than 0.7 M urea under constant stirring wherein the protein concentration during refolding is maintained at greater than 500 μg/ml to provide a proEP-B2 enzyme composition capable of self-activation at acidic pH.
2. The method of claim 1 , wherein the fermentation reaction is in a volume greater than 10 liters.
3. The method of claim 1 , wherein the fermentation reaction is in a volume greater than 100 liters.
4. The method of claim 2 , wherein the microbial host comprises polynucleotide sequences encoding proEP-B2, in the absence of the native signal sequence, operably linked to an inducible promoter.
5. The method of claim 4 , wherein the microbial host is E. coli.
6. The method of claim 5 , wherein the proEB-B2 enzyme is harvested after inducing transcription from said promoter for 2 to 5 hours, at a temperature of from 28° C. to about 32° C.
7. The method of claim 6 , wherein the proEB-B2 enzyme is harvested after inducing transcription from said promoter for about 3 hours.
8. The method of claim 4 , wherein the polynucleotide sequences encoding proEP-B2 comprise at least one affinity tag to facilitate purification.
9. The method of claim 8 , wherein the affinity tag is a terminal hexa-histidine sequence.
10. The method of claim 9 , wherein the proEP-B2 enzyme is purified by nickel affinity chromatography.
11. The method of claim 1 , wherein the proEP-B2 enzyme concentration during refolding is maintained at less than 1 mg/ml.
12. The method of claim 11 , further comprising purification of the proEP-B2 enzyme by a combination of two or more steps of selective precipitation, reverse phase chromatography, gel exclusion chromatography, and ion exchange chromatography.
13. The method of claim 11 , further comprising lyophilizing the proEP-B2 enzyme composition.
14. The method of claim 11 , wherein the proEP-B2 enzyme composition has a specific activity of at least about 500 U/mg when activated at acidic pH, where 1 unit is defined as 1 μM p-nitroaniline released per minute from a chromogenic substrate CBz-Phe-Arg-pNA at room temperature.
15. The method of claim 14 , wherein the proEP-B2 enzyme composition has a specific activity of at least 1000 U/mg when activated at acidic pH.
16. A method of producing a self-activating active cysteine endoprotease B, isoform 2 proenzyme (proEP-B2) from cytosolic inclusion bodies, the method comprising:
solubilizing the cytoplasmic inclusion bodies by mechanical dissociation in urea at a concentration of at least 7M to produce solubilized, denatured, proEP-B2, purifying said solubilized, denatured, proEP-B2; and
refolding said purified, solubilized, proEP-B2, by rapidly diluting the solubilized proEP-B2 into buffer comprising not more than 0.7 M urea, wherein the protein concentration is maintained at 500 μg/ml concentration and less than 1 mg/ml to provide a proEP-B2 enzyme composition capable of self-activation at acidic pH.
17. The method of claim 16 , wherein the cytoplasmic inclusion bodies are produced in a fermentation reaction with a microbial host comprising polynucleotide sequences encoding proEP-B2, in the absence of the native signal sequence, operably linked to an inducible promoter.
18. The method of claim 17 , wherein the microbial host is E. coli.
19. The method of claim 17 , wherein the polynucleotide sequences encoding proEP-B2 comprise at least one affinity tag to facilitate purification.
20. The method of claim 19 , wherein the affinity tag is a terminal hexa-histidine sequence.
21. The method of claim 20 , wherein the proEP-B2 enzyme is purified by nickel affinity chromatography.
22. The method of claim 16 , further comprising purification of the proEP-B2 enzyme by a combination of two or more steps of selective precipitation, reverse phase chromatography, gel exclusion chromatography, and ion exchange chromatography.
23. The method of claim 16 , further comprising lyophilizing the proEP-B2 enzyme composition.
24. The method of claim 16 , wherein the refolded proEP-B2 enzyme is formulated in a composition that when activated at acidic pH has a specific activity of at least 500 U/mg, where 1 unit is defined as 1 μM p-nitroaniline released per minute from a chromogenic substrate CBz-Phe-Arg-pNA at room temperature.
25. The method of claim 24 , wherein the proEP-B2 enzyme composition has a specific activity of at least 1000 U/mg when activated at acidic pH.