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- Title
Advanced purification platform using circularly permuted caspase‐2 for affinity fusion‐tag removal to produce native fibroblast growth factor 2.
- Authors
Lingg, Nico; Cserjan‐Puschmann, Monika; Fischer, Andreas; Engele, Petra; Kröß, Christina; Schneider, Rainer; Brocard, Cécile; Berkemeyer, Matthias; Striedner, Gerald; Jungbauer, Alois
- Abstract
BACKGROUND: Recombinant proteins produced for use as biopharmaceuticals need to harbor their native N‐terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion‐tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N‐terminus. RESULTS: Human fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed‐batch cultivations. The protein of interest (POI) carried an N‐terminal affinity fusion‐tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion‐tag with a circularly permuted human caspase‐2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N‐terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 μg mL−1 dsDNA and ~ 1000 EU mL−1 endotoxin. Only a small number of E. coli host cell proteins were co‐purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off‐target cleavage could be observed. CONCLUSION: Our findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusion‐protein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2‐based platform fusion protein process (CASPON technology) purification platform. © 2021 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
- Subjects
FIBROBLAST growth factor 2; SOCIETY of Chemical Industry (Great Britain); JOHN Wiley &; Sons Inc.; CASPASES; CHIMERIC proteins; RECOMBINANT proteins; CHEMICAL processes; FIBROBLAST growth factors
- Publication
Journal of Chemical Technology & Biotechnology, 2021, Vol 96, Issue 6, p1515
- ISSN
0268-2575
- Publication type
Article
- DOI
10.1002/jctb.6666