Flexible linkers

[Sean Speese-2]

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This is a tad bit off topic, but I was wondering if people would be
willing to share their experience with flexible linkers used when making
fusions of your protein of interest to fluorescent proteins.  What are some
sequences that seem to work well?  This will likely be empirical, but still
useful information for me in deciding what linkers to use in the future.  

Thanks,
   
   Sean Speese

[Michael Schell]

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We've mostly used GGSGS, which is flexible with a bit of hydrophililc character.  We did a comparison whereby we compared this linker to no linker whatsoever.  The EGFP fluorescence was far brighter with the linker, presumably because the EGFP folded better.

However, there have been situations where a stiff or bulky linker (DPPVAT,  the inker formed when cloning into EGFPN1; or YSDLELKF from pEGFPC3)  turned out to be preferable.  We assume that, in these cases,  the kinks created by the dual prolines  or else aromatic side groups push the GFP away from the protein it is fused to, and minimizes some sort of undesirable steric hindrance.  In lieu of structural info, a certain amount of guesswork is unavoidable.

Mike

 
Michael J. Schell, Ph.D., CIV, USUHS
Assist. Professor
Dept. of Pharmacology
Uniformed Services University
4301 Jones Bridge Rd.
Bethesda, MD  20814-3220
tel:  [phone]
[hidden email]
>>> Sean Speese <[hidden email]> 10/04/10 2:08 PM >>>
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This is a tad bit off topic, but I was wondering if people would be
willing to share their experience with flexible linkers used when making
fusions of your protein of interest to fluorescent proteins.  What are some
sequences that seem to work well?  This will likely be empirical, but still
useful information for me in deciding what linkers to use in the future.  

Thanks,
   
   Sean Speese

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[Vitaly Boyko]

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Hi Sean,
 
though I haven't published it, I compared a number of variants with the best
linker has been described in the 2008 paper pasted below (Mat&Methods section).
Luckily my protein was still functional when fused to GFP/YFP, and I had
designed a simple test for the protein functionality, which is in part described
in the 2000 paper (Fig. 1).
 
1. Chukkapalli V., I. B. Hogue, V. Boyko, W.S. Hu, and A. Ono. 2008. Interaction
between HIV-1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is
essential for efficient Gag-membrane binding. J. Virol. 82(5):2405-17.

 
2. Boyko, V., J. Ferralli, J. Ashby, P. Schellenbaum, and M. Heinlein. 2000.
Function of microtubules in intercellular transport of plant virus RNA. Nature
Cell Biol. 2:826-832. This research paper described several
temperature-sensitive mutants of the movement protein

If you have any questions, please let me know.
 
Good luck,
 
Vitaly
301-515-7833




________________________________
From: Sean Speese <[hidden email]>
To: [hidden email]
Sent: Mon, October 4, 2010 2:07:55 PM
Subject: Flexible linkers

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This is a tad bit off topic, but I was wondering if people would be
willing to share their experience with flexible linkers used when making
fusions of your protein of interest to fluorescent proteins.  What are some
sequences that seem to work well?  This will likely be empirical, but still
useful information for me in deciding what linkers to use in the future. 

Thanks,
 
  Sean Speese