Self-activating factor X derivative fused to the C-terminus of a cellulose-binding module: Production and properties.
In this work, a new derivative of FX was engineered. It comprises a cellulose-binding module (CBM) fused to the N-terminus of the truncated light chain (E2FX) of FX and a hexahistidine tag (H6) fused to the C-terminus of the heavy chain. The sequence LTR at the site of cleavage of the activation peptide from the N-terminus of the heavy chain is changed to IEGR to render the derivative self-activating. However, N-linked glycans on the CBM of the derivative blocked its binding to cellulose and those on the activation peptide slowed its activation. Therefore, the sites of N-linked glycosylation on the CBM and on the activation peptide were eliminated by mutation. The final derivative can be produced in good yield by cultured mammalian cells. It is purified easily with Ni(2+)-agarose, it is self-activating, and it can be immobilized on cellulose. When immobilized on a column of cellulose beads, the activated derivative retains approximately 80% of its initial activity after 30 days of continuous hydrolysis of a fusion protein substrate. Under these conditions of operation, the effective substrate:enzyme ratio is >10(4).
Kwan
Emily
E
Department of Microbiology and Immunology, University of British Columbia, Vancouver BC, Canada.
Guarna
M Marta
MM
Boraston
Alisdair B
AB
Gilkes
Neil R
NR
Haynes
Charles A
CA
Kilburn
Douglas G
DG
Warren
R Antony J
RA
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D018914:Cells, Immobilized; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D005170:Factor X / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012685:Sepharose; D013045:Species Specificity
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D005170:Factor X; D002482:Cellulose; D012685:Sepharose