Matrix-assisted refolding of autoprotease fusion proteins on an ion exchange column.
Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N(pro) autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO(3)(-), UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris-HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO(3)(-), refolding could be performed at column loadings of 50mg fusion protein/ml gel, resulting in a final eluate concentration of around 10-15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.
Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.
Elisabeth
E
Schmoeger
Eva
E
Berger
Alexandru
A
Trefilov
Alois
A
Jungbauer
Rainer
R
Hahn
D000327:Adsorption; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D007474:Ion Exchange; D007700:Kinetics; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature
D010455:Peptides; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases