title: Immobilization of recombinant heparinase I fused to cellulose-binding domain.
abstract: Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.
authors.affiliation: The Kennedy Leigh Centre for Horticultural Research and The Otto Warburg Center for Agricultural Biotechnology, The Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
authors.forename: E
authors.initials: E
authors.lastname: Shpigel
authors.forename: A
authors.initials: A
authors.lastname: Goldlust
authors.forename: G
authors.initials: G
authors.lastname: Efroni
authors.forename: A
authors.initials: A
authors.lastname: Avraham
authors.forename: A
authors.initials: A
authors.lastname: Eshel
authors.forename: M
authors.initials: M
authors.lastname: Dekel
authors.forename: O
authors.initials: O
authors.lastname: Shoseyov
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D019149:Bioreactors; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004800:Enzymes, Immobilized / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006493:Heparin; D019763:Heparin Lyase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose; D006493:Heparin; D019763:Heparin Lyase