Immobilisation of bovine enterokinase and application of the immobilised enzyme in fusion protein cleavage.
Two immobilisation methods for enterokinase were developed, which yielded high remaining activities for the cleavage of the fusion protein MUC1-IgG Fc. Different carrier materials were compared regarding remaining enzyme activity and storage stability. Immobilisation procedures involving support material activation using glutardialdehyde were found to result in low remaining activities. Applying less aggressive activation procedures, remaining activities of approximately 60% were received when immobilising enterokinase on either Estapor paramagnetic microspheres or hexamethylamino Sepabeads. In case of hexamethylamino Sepabeads we were able to increase the half-life time 4.3-fold at 23 degrees C and 3.8-fold at 4 degrees C compared to the free enzyme at the same temperatures. By immobilising the biocatalyst the downstream process is simplified allowing the easy removal of the enzyme from the reaction mixture. The immobilised enterokinase cleaves the fusion protein MUC1-IgG Fc in at least two repeated batches, proving the efficiency of the immobilisation method and the reusability of the biocatalyst.
Kubitzki
Tina
T
Institute of Biotechnology 2, Research Centre Juelich GmbH, 52425 Jülich, Germany.
Noll
Thomas
T
Lütz
Stephan
S
D000818:Animals; D001671:Biochemistry / Q000379:methods; D001709:Biotechnology / Q000379:methods; D002417:Cattle; D004765:Enteropeptidase / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005976:Glutaral / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D008280:Magnetics; D008863:Microspheres; D008956:Models, Chemical; D018396:Mucin-1 / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D018396:Mucin-1; D011993:Recombinant Fusion Proteins; D004765:Enteropeptidase; D005976:Glutaral