Hi Folks,

First of all, thanks to all the people that responded. Many of you  
asked me for a summary of responses. So, below is a concise summary  
containing mainly the references that people pointed me to:

Cheers,
Raji

-----------------------------------
ORIGINAL POST:
Hi People,

Could anyone point me to successful examples for two unrelated  
proteins that have been stitched together into one single polypeptide  
chain with flexible amino acids to create a functional chimera that  
was subsequently crystallized. I've looked up a few.

I am particularly interested in understanding all the important  
considerations while designing a flexible linker even though many of  
these factors might be case dependent might be variable, obvious and  
commonsensical. Either way, I'd like to hear what folks have to say.

Thanks very much.
Raji


-----------------------------------
SUMMARY OF RESPONSES:

1. VAMP7 longin domain (a SNARE protein) and its binding partner, Hrb.
Cell. 2008 Sep 5;134(5):817-27
http://www.pubmedcentral.nih.gov/articlerender.fcgi? 
tool=pubmed&pubmedid=18775314
- Lauren Jackson

2. To look at lysozyme engineering in the case of the GPCR. Also, to  
look at Bostjan Kobe's papers, who has been advocating  
crystallization of MBP and GST fusion proteins.
- Boaz Shaanan

3. To check out the structure and method paper for a TrxA-RRM domain  
fusion. The design of the linker is discussed in the method paper.
Dimerization and protein binding specificity of the U2AF homology  
motif of the splicing factor Puf60.
Corsini L, Hothorn M, Stier G, Rybin V, Scheffzek K, Gibson TJ,  
Sattler M.
J Biol Chem. 2009 Jan 2;284(1):630-9. Epub 2008 Oct 29.
Thioredoxin as a fusion tag for carrier-driven crystallization.
Corsini L, Hothorn M, Scheffzek K, Sattler M, Stier G.
Protein Sci. 2008 Dec;17(12):2070-9.
-Michael Hothorn

4. Panne et al. Cell. 2007 Jun 15;129(6):1111-23.
Nature. 2008 May 22;453(7194):489-4.
- Jayakrishnan Nandakumar

5. MBP-linker-Target. It was one of our targets in Berkeley  
Structural Genomics Center. The linker was Gly4Ser. Both, the target  
with His-tag and the fusion one crystallized with similar resolution  
of around 2.3A
- Vaheh Oganesyan

6. One example is by David Owen's group in Cambridge.
Molecular basis for the sorting of the SNARE VAMP7 into endocytic  
clathrin-coated vesicles by the ArfGAP Hrb.
Pryor PR, Jackson L, Gray SR, Edeling MA, Thompson A, Sanderson CM,  
Evans PR, Owen DJ, Luzio JP.
Cell. 2008 Sep 5;134(5):817-27.
- Bret Collins

7. http://www.pubmedcentral.nih.gov/articlerender.fcgi? 
tool=pubmed&pubmedid=15159535
- Richard Kingston

8. Example of a domain-swapped chimera
Biochem. J. (2006) 393, 767–777
- Selvaraj

9. See 1axk
- Juergen-Joachim Mueller

10. James Stroud already posted to the BB.

11. What about single-chain Fv contructs of antibodies? PDB accession  
code 1DZB, for example.
- Joe

12. Examples: GPCR's fused with T4 lysozyme. Fusion of cytochrome  
oxidase soluble part to Lac permease (Ron Kaback lab).
- Vinothkumar

13. Structural basis for the attachment of a paramyxoviral polymerase  
to its template.
Kingston RL, Hamel DJ, Gay LS, Dahlquist FW, Matthews BW.
Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8301-6.
- Anthony Addlagatta

14. Panne, D., Maniatis, T., and Harrison, S. C. (2004) Crystal  
structure of ATF-2/c-Jun and IRF-3 bound to the interferon-beta  
enhancer, EMBO J 23, 4384-4393.
Panne D, Maniatis T, Harrison SC, (2007) An atomic model of the  
interferon-beta enhanceosome.  Cell. 2007 Jun 15;129(6):1111-23.
- Tiancen/Tony Hu

15. Smyth et al.,   Protein Sci (2003), 12, 1313-1322.
- Ramanathan Natesh

16. Suggestion to use protease cleavage sites as linkers.
- Artem Evdokimov

17. Smyth DR, Mrozkiewicz MK, McGrath WJ, Listwan P, Kobe B. Crystal  
structures of
fusion proteins with large-affinity tags. Protein Sci. 2003 Jul;12(7): 
1313-22.
- Bostjan Kobe