Sugiura T, Takamatsu H, Kudo A, Amann E
Laboratory of Molecular Biology, Hoechst Japan Limited, Saitama.
Protein Expr Purif. 1995 Jun;6(3):305-11. doi: 10.1006/prep.1995.1040.
Osteoblast-specific factor 2 (OSF-2) is a approximately 90-kDa protein selectively expressed in bone. OSF-2 cDNA was recently isolated from mouse and human cDNA libraries and shows limited sequence homology with fasciclin I, a cell adhesion protein expressed in insect nerve cells. Here we describe the expression of recombinant murine OSF-2 (rmOSF-2) in a baculovirus/insect cell system. Western blotting analysis employing polyclonal antiserum raised against a C-terminal synthetic OSF-2 peptide detected a protein of approximately 90-kDa as early as 2 days after infection of Sf9 cells with the recombinant virus. Tunicamycin treatment of infected cells resulted in a mobility shift of OSF-2 (approximately 90-kDa band) on Western blots. N-Glycanase digestion resulted in the same mobility shift of OSF-2, indicating that rmOSF-2 expressed in insect cells is N-glycosylated. However, OSF-2 was insensitive to endoglycosidase H digestion while a major fraction of this protein had affinity for concanavalin A. Finally, it was demonstrated that rmOSF-2 was able to bind to heparin. This finding suggests that OSF-2 might be associated with the bone extracellular matrix after secretion by osteoblasts and participate in cell adhesion and/or cell communication. The establishment of the baculovirus expression system with a high productivity of recombinant OSF-2 (around 40 micrograms/ml at maximum) and its heparin binding properties should allow us to obtain large amounts of rmOSF-2.
成骨细胞特异性因子2(OSF-2)是一种在骨中选择性表达的约90 kDa的蛋白质。OSF-2 cDNA最近从小鼠和人cDNA文库中分离得到,与在昆虫神经细胞中表达的细胞粘附蛋白fasciclin I具有有限的序列同源性。在此,我们描述了重组鼠OSF-2(rmOSF-2)在杆状病毒/昆虫细胞系统中的表达。用针对C端合成OSF-2肽产生的多克隆抗血清进行的蛋白质印迹分析,早在重组病毒感染Sf9细胞2天后就检测到了一种约90 kDa的蛋白质。用衣霉素处理感染细胞导致蛋白质印迹上OSF-2(约90 kDa条带)的迁移率发生改变。N-糖苷酶消化导致OSF-2出现相同的迁移率改变,表明在昆虫细胞中表达的rmOSF-2是N-糖基化的。然而,OSF-2对内切糖苷酶H消化不敏感,而该蛋白的大部分对伴刀豆球蛋白A具有亲和力。最后,证明rmOSF-2能够结合肝素。这一发现表明,OSF-2在成骨细胞分泌后可能与骨细胞外基质相关,并参与细胞粘附和/或细胞通讯。具有高产量重组OSF-2(最大约40微克/毫升)的杆状病毒表达系统的建立及其肝素结合特性应该使我们能够获得大量的rmOSF-2。