In a different experiment, we modified the chromosomal loci of the  
spore coat protein genes for SP85 and SP65 with GFP-coding sequences  
to express normal levels of the fusion proteins with the correct  
timing. GFP fluoresced better after exocytosis during sporulation than  
before, and we found that growing the amoebae on bacteria minimized  
the problem of proteolytic cleavage (T. Metcalf et al., 2007). Good  
luck.  -Chris


On Apr 14, 2009, at 2:18 PM, Brzostowski, Joseph (NIH/NIAID) [E] wrote:

> Hi Tony,
>
> Type I proteins can be cleaved by proteases, which can account for  
> your low signal. I placed an N-term FLAG-tag in a type I  
> transmembrane protein to analyze this phenomenology (sorry not  
> published). Check your growth media (or even starvation media) by  
> western to see if the protein is being released from the membrane -  
> you might need to concentrate the media before running the gel.
>
> Best,
>
> Joe
>
>
> On 4/14/09 2:26 PM, "Anthony Kowal" <[log in to unmask] 
> > wrote:
>
>
> Hello All,
>
> I was wondering if anyone knows of any (or has heard of any) plasma  
> membrane proteins which have been tagged at the N-terminus with  
> GFP.  I realize that the GFP coding sequence would have to be placed  
> between the signal peptide sequence and the N-terminus of the  
> protein, so that the fusion protein is targeted correctly to the  
> secretory pathway.  I know of, and have used, Transmembrane proteins  
> which have been tagged at the C-terminus with GFP, and both proteins  
> are still functional.  Would one expect GFP to fluoresce  
> extracellularly?
>
> I have actually tried to tag a multipass transmembrane protein with  
> GFP at the N-terminus (so it is extracullular), and although I  
> observe GFP fluorescence, it is much weaker than when GFP is  
> intracellular.  If I supplement the imaging media with ~20mM reduced  
> glutathione, the fluorescence is more pronouced, but still weak.   
> The DNA sequence was verified, and all of the known amino acids for  
> GFP to fold correctly and fluoresce are present.  The protein of  
> interest is still functional.
>
> Any thoughts/suggestions/ideas are very much appreciated.
>
> Thank you for your time.
>
> Best -
>
> Tony
>
>
> --
> Joseph Brzostowski, Ph.D.
> LIG Imaging Facility, Head
> National Institutes of Health/NIAID
> 12441 Parklawn Dr. Twin II/Rm. 201
> Rockville, MD 20852
>
> Mobile: 240-599-6364
> Cu Wire: 301-443-2967
> Fax: 301-402-0259
>
> [log in to unmask]