Startseite
English
Erweiterte Suche
Durchblättern nach ...
Publikationen im Überblick
Ansprechpartner
Hilfe
DOI
PMC
 |
|
Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
A novel expression and purification system for the production of enzymatic and biologically active human granzyme B.
J. Immunol. Methods 371, 8-17 (2011)
The serine protease granzyme B (grB) has previously been shown to induce perforin-independent apoptosis in membrane Hsp70 positive tumor cells in a number of in vitro experimental systems. Ongoing studies that are investigating the in vivo relevance of these findings by assessing the therapeutic potential of grB in a human xenograft tumor mouse model required the development of an expression system for the production of high yields of enzymatic and biologically active human grB. In order to maintain potentially important posttranslational modifications that occur in mammalian cells, human embryonal kidney cells (HEK293) were stably transfected with human grB. The HEK293 host cells were protected from apoptotic cell death by fusing an inactivation site coupled to a (His)(6) tag to the gene sequence of GrB. Inactive grB which was actively released from HEK293 cells by insertion of a Igκ leader sequence was purified on a nickel column utilizing the (His)(6) tag. After enterokinase digestion and heparin affinity chromatography, high yields of enzymatic and biologically active human grB were obtained. The perforin-independent interaction of grB with membrane Hsp70 positive tumor cells appeared to be associated with mammalian glycosylation and mediated by the oligosaccharide moiety of neuraminic acid (NANA).
Altmetric
Weitere Metriken?
Zusatzinfos bearbeiten
[➜Einloggen]
Publikationstyp
Artikel: Journalartikel
Dokumenttyp
Wissenschaftlicher Artikel
Schlagwörter
Granzyme B; Purification; Enterokinase; Activity; HEK293 cells; Neuraminic acid
ISSN (print) / ISBN
0022-1759
e-ISSN
1872-7905
Zeitschrift
Journal of Immunological Methods
Quellenangaben
Band: 371,
Heft: 1-2,
Seiten: 8-17
Verlag
Elsevier
Nichtpatentliteratur
Publikationen
Begutachtungsstatus
Peer reviewed
Institut(e)
CCG Innate Immunity in Tumor Biology (PATH-KTB)