in vitro glycosylation

David Ashford da5 at york.ac.uk
Tue Apr 15 11:51:38 EST 1997


Dear Steve,

I don't have any experience of the Promega kit you mention but I can help 
on the N-glycosylation stuff.

All eukaryotes seem to share the early steps of N-glycosylation. They add a 
preformed glycan to the sequence Asn-Xaa-Ser/Thr. This is done 
co-translationally in the ER. So if your protein has an appropriate 
sequence the canine microsome preparation should N-glycosylate it. There 
are differences between the mature structures on animal, yeast and plant 
glycoproteins but these are due to processing events and additions 
catalysed primarily by Golgi enzymes.

I think your experiment should work as you describe it. To check that any 
shift in mol. wt. id due to N-glycosylation you could check for 
susceptibility to Endo H. But that's another story.

Dave Ashford

*************************************************************************
   Dr David A. Ashford                                                  
   Glycobiology: Research & Analytical                                
   Department of Biology                                                
   University of York                         Tel: (44)(0)1904 434391   
   P.O. Box 373                               Fax: (44)(0)1904 434312   
   York  YO1 5YW                              Email:  da5 at york.ac.uk    
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On 15 Apr 1997 14:26:05 GMT Steve Dunn wrote:

> From: Steve Dunn <dunnsm at bbsrc.ac.uk>
> Date: 15 Apr 1997 14:26:05 GMT
> Subject: in vitro glycosylation
> To: "bionet.glycosci mail newsgroup" <bionet-news at dl.ac.uk>
> 
> Hi!
> I'm wondering whether anyone's familiar with Promega's T7 TNT quick
> kit for in vitro transcription/translation.  I wish to use it in
> conjunction with their canine microsomes to investigate possible
> glycosylation/processing of my gene which encodes a membrane protein.
> The catch! My protein is from a plant not mammalian. So, if I see any
> glycosylation, is it likely to be relevant and a good indication of
> what might be occurring in the plant ie. in vivo (I accept the nature of
> the glycosyl moiety is likely to be different, it's just the occurrence
> I'm conserved with). I guess I'm really asking whether glycosylation
> recognition sites are conserved between eucaryotes. Can anybody shed
> some light on this ?
> Many thanks for any enlightening comments,
> Steve
> 
> dunnsm at bbsrc.ac.uk






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