Hi,
Yes �C it’s quite likely that your protein is glycosylated. No worries, it’s not a death knell �C in fact glycosylation can be interpreted as a good sign :-) Crystallization: yes, this may be an issue. On the other hand it could also be not a problem. Try crystallizing the protein as-is, but be prepared to strip the CHO off �C either enzymatically, or via mutagenesis. Be prepared for changes in expression level of mutants �C can be same expression levels as native protein but unfortunately in many cases expression goes down or even away completely. You could *try* inhibiting glycosylation via chemicals but that could end up being too expensive - also inhibitor-treated cells tend to be quite sick. Artem “Nothing is built on stone; all is built on sand, but we must build as if the sand were stone” Jorge Luis Borges _____ From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of ruheng Sent: Thursday, September 03, 2009 3:39 AM To: [email protected] Subject: [ccp4bb] problem in expression of glycoproteins in baculovirus expression system Dear all, Recently I am secretely expressing an ectodomain of a human membrane protein in insect cells. The expression level is moderately high but the target band in the SDS-PAGE gel seems smearing and this is cosistent with the western blot results. Does it result from the heterogeneous glycosylation of the protein? My protein contains quite a lot of N and O linked polysaccharide chains and they are important for the function of the protein. Does the heterogeneously glycosylated protein affect the crystallization? Does anyone have the experience of crystallizing such kind of glycoprotein? Thanks in advance! Ru Heng _____ 搜索本应是快乐的，不是么? 快乐搜索，有问必应！微软隆重推出! <http://bing.com.cn?FORM=M00HCN&Publ=WLHMTAG&Crea=TEXT_Search_Where_You_Are_ 1X1> 立即试用！
