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
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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
