Intasai Nutjeera, Arooncharus Pramoon, Kasinrerk Watchara, Tayapiwatana Chatchai
Department of Clinical Microscopy, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
Protein Expr Purif. 2003 Dec;32(2):323-31. doi: 10.1016/j.pep.2003.08.019.
Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfully increased when growing the transformed TG1 at 25 degrees C with IPTG-stimulation. In addition to temperature and IPTG-stimulation, the VCSM13 helper phage infection-period particularly affected the insertion of CD147Ex into phage progeny. By sandwich ELISA, incorporation of the CD147Ex into phage particle was confirmed. The correct size of the CD147Ex-gpVIII fusion protein at 28kDa was demonstrated by Western immunoblotting. Multivalent display of CD147Ex on phage particles will be valuable in discovering its ligand partner.
当培养条件得到调控时，可实现高密度展示CD147胞外域（CD147Ex）的VCSM13噬菌体的产生。构建了一个表达CD147Ex的噬菌粒，并用于在TG1大肠杆菌中产生噬菌体展示的CD147Ex gpVIII融合蛋白。当在25摄氏度下用IPTG诱导培养转化后的TG1时，通过gpVIII成功增加了CD147Ex的展示。除了温度和IPTG诱导外，VCSM13辅助噬菌体感染期对CD147Ex插入噬菌体后代的影响尤为显著。通过夹心ELISA法，证实了CD147Ex掺入噬菌体颗粒中。通过蛋白质免疫印迹法证实了CD147Ex-gpVIII融合蛋白的正确大小为28 kDa。噬菌体颗粒上CD147Ex的多价展示对于发现其配体伴侣将具有重要价值。
