An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.
Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.
Forsberg
G
G
Kabi Pharmacia KabiGen, Stockholm, Sweden.
Baastrup
B
B
Rondahl
H
H
Holmgren
E
E
Pohl
G
G
Hartmanis
M
M
Lake
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000378:metabolism; D004800:Enzymes, Immobilized; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D000596:Amino Acids; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C058957:insulin-like growth factor 1, des-(1-3)-; D007334:Insulin-Like Growth Factor I; D013381:Subtilisins; D013917:Thrombin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D004765:Enteropeptidase