In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant.
In order to show site-specific cleavage of fusion proteins with an engineered soluble yscF variant, we have constructed a fusion gene encoding eglinC from Hirudo medicinalis and the cellulose-binding domain from the Cellulomonas fimi exoglucanase (Cex). The two fusion partners were separated by a Lys-Arg-containing recognition sequence for the yeast endoprotease yscF (Kex2). The fusion protein (eglinC-Cex) was expressed intracellularly in Saccharomyces cerevisiae. After disruption of the cells eglin-C-Cex was shown to bind to cellulose when present in total crude cell lysates. This step efficiently removed the majority of contaminating host proteins. While immobilized on cellulose, eglin-C-Cex could be cleaved into the expected fragments. Upon cleavage the eglinC part was released from the cellulose, while the purification tag, i.e. the cellulose binding domain, stayed bound to the cellulose matrix.
Seeboth
P G
PG
Ciba-Geigy Ltd., Biotechnology Department, Basel, Switzerland.
Warren
R A
RA
Heim
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism*; D003001:Cloning, Molecular; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D043484:Proprotein Convertases; D011485:Protein Binding; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism; D015843:Serpins; D013381:Subtilisins
D004800:Enzymes, Immobilized; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C028656:eglin proteinase inhibitors; D002482:Cellulose; D005959:Glucosidases; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae