Immobilization of recombinant heparinase I fused to cellulose-binding domain

Etai Shpigel, Arie Goldlust, Gilat Efroni, Amos Avraham, Adi Eshel, Mara Dekel, Oded Shoseyov*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

68 Scopus citations

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-Hepl and Hepl-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-Hepl-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-Hepl, and cellulose-immobilized CBD-Hepl, were compared. The profiles obtained by free heparinase I and CBD-Hepl were indistinguishable, however, immobilized CBD-Hepl 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.

Original languageAmerican English
Pages (from-to)17-23
Number of pages7
JournalBiotechnology and Bioengineering
Volume65
Issue number1
DOIs
StatePublished - 5 Oct 1999

Keywords

  • Bioreactor
  • Cellulose-binding domain
  • Fusion protein
  • Immobilization
  • Low-molecular weight heparin

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