sugar binding column and deglycosylation

Steven Pirie-Shepherd srps at galactose.mc.duke.edu
Thu Sep 28 07:53:39 EST 1995


James Fethiere (James.Fethiere at BRI.NRC.CA) wrote:
: Hello all,

: I have expressed a glycoprotein in sf9 cells, and it seems to be highly
: sialated (according to sensitivity to neuraminidase).  I am now looking
: for a lectin column with a higher specificity and affinity for sialated
: carbohydrates as an extra step in my purification scheme.  Any suggestions??


I would try SNA (specific for a(2-6) SA). This works well for terminal 
sialic acids in this linkage, which yours are likely to be given your CHO 
seems to be an N-linked structure.



: The protein migrates as 4 distinct bands on an SDS-PAGE gel.  The highest MW
: band is the first coming out of the Q-sepharose column, and is also the 
: one that collapses upon treatment with neuraminidase.  However subsequent
: treatment of the sample with endo-F did not yield further processing of
: the carbohydrates while treatment with N-glycanase/Endo-F alone provoked
: collapsing of all 4 bands to a single one.  I was wondering why Endo-F
: did not have any effect either by itself or after neuraminidase.  The enzyme
: was not too old, and an excess of it was added ( > 2U/mg protein), so ...

Endo F cleaves between the two GlcNAc moieties whilst PNGaseF 
(N-glycanase) cleaves between Asp and sugar. It is a much better enzyme 
for this sort of thing.

SNA is Sambucus Nigra agglutinin. The buffer I use is 50mM Tris/1mM 
CaCl2/1mM MgCl2, pH 7.4 (HCl). I elute bound material using this buffer 
with 0.1M Lactose (odd choice but it works, neat sialic acid is far too 
expensive to use as a competeing ligand. SNA can be obtained from Boehringer.

Hope this helps.

--


__________________________________________________
Steven Pirie-Shepherd		       		
srps at galactose.mc.duke.edu	        
"Insert your own pithy phrase just about here!"



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