title: Purification of human interleukin-2 using the cellulose-binding domain of a prokaryotic cellulase.
abstract: Engineering gene fusions which introduce an affinity tag linked to the target polypeptide by a specific protease cleavage site is widely used to facilitate recombinant protein purification. A fusion protein CBDAPT-IL-2, comprised of the cellulose-binding domain (CBD) and Pro-Thr (PT) rich linker of the Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) and a factor Xa cleavage sequence (IleGluGlyArg) fused to the N terminus of human interleukin-2, was produced in Escherichia coli, Streptomyces lividans and mammalian COS cells. CBDAPT-IL-2, secreted from S. lividans or COS cells or recovered from the insoluble fraction of E. coli, could be purified by adsorption on cellulose. The intact fusion protein adsorbed to cellulose was hydrolyzed in situ with factor Xa to release active interleukin-2.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Ong
authors.forename: J B
authors.initials: JB
authors.lastname: Alimonti
authors.forename: J M
authors.initials: JM
authors.lastname: Greenwood
authors.forename: R C
authors.initials: RC
authors.lastname: Miller
authors.forename: R A
authors.initials: RA
authors.lastname: Warren
authors.forename: D G
authors.initials: DG
authors.lastname: Kilburn
mesh_terms: D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007376:Interleukin-2 / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics
chemical_list: D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D002480:Cellulase; D015951:Factor Xa