I am making a fusion protein with E. coli enzyme ilvD and the fluorescent protein mNeonGreen and I am trying to find more information regarding what the linker sequence between the proteins should look like.
One sequence I am considering is the [Gly(4)Ser]x3 sequence, but I am interested in more options.
I should also say that I am only interested in expression level of ilvD, so it is not important to me that ilvD remain catalytically active, only that fusion protein transcription/translation is efficient and that mNeonGreen can efficiently fold/fluoresce.
Does anyone have any experience with fluorescent fusion protein linkers that would be pertinent?
Thank you very much!
Use sth flexible and hydrophilic like the sequence you're suggesting. My personal favourite is (GSSG)2 or 3. Maybe put a prolin in, too, to force a break in the secondary structure. In order not to compromise expression levels, have a look on proper codon usage (i.e. avoid rare ones).
(GSSG) 2 or 3 is a good choice. I have sandwiched an E coli protein between two FPs and the expression rate was pretty well even w/o any linker or codon usage. Of course, codon usage will accelerate the expression machinery.
We've used GGSGGS for a couple of different applications (split GFP and for single molecule studies [DOI: 10.1002/pro.2952]) and it's been fine. Expression is fine and no issues with proteolysis in the linker.
@Mark: need to be afraid of any proteases which like to nibble on that?
@Wolfgang. None that I know of but thought that I'd mention that it hasn't been an issue.
The GGGGS linker is really a classic. From as little as one repeat have been used. Two or three repeats is often used, but examples with greater number of repeats were reported. Depending on the expression system, you want to avoid sequence that could represent potential protease recognition sites. If you are not interested in detecting binary interactions (using the linker as a molecular ruler), the length of the linker is not very important. See the refs for examples of how the length of the linker was used to extract relevant information from detected binary interactions:
1: Remy I, Wilson IA, Michnick SW. Erythropoietin receptor activation by a
ligand-induced conformation change. Science. 1999 Feb 12;283(5404):990-3. PubMed
PMID: 9974393.
2: Tarassov K, Messier V, Landry CR, Radinovic S, Serna Molina MM, Shames I,
Malitskaya Y, Vogel J, Bussey H, Michnick SW. An in vivo map of the yeast protein
interactome. Science. 2008 Jun 13;320(5882):1465-70. doi:
10.1126/science.1153878. Epub 2008 May 8. PubMed PMID: 18467557.
3: Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux KJ. An
improved smaller biotin ligase for BioID proximity labeling. Mol Biol Cell. 2016
Apr 15;27(8):1188-96. doi: 10.1091/mbc.E15-12-0844. Epub 2016 Feb 24. PubMed
PMID: 26912792; PubMed Central PMCID: PMC4831873.
Hi all,
Which codons would you use for G and S for protein expression in Ecoli (BL21)?
Thank you.
N
- Badges
- Science topic