Hello, I am preparing a fusion protein with mentioned sequences and I have doubts about the type of linker to use. I have seen in the literature that sometimes flexible linkers of this type of sequence are used: (Gly-Gly-Gly-Gly-Ser)n.but when I use flexible linkers including Gly-Gly-Gly-Gly-Ser , GGGGGG , or every linker with Gly, discotope tools predict linkers as a antigenic domain.
(DiscoTope: Structure-based Antibody Prediction)
http://tools.immuneepitope.org/stools/discotope/discotope.do
I have used a(EAAAK)3 as Rigid linker
As suggested by George and Heringa (2002), many natural
linkers exhibited α-helical structures. The α-helical structure was
rigid and stable, with intra-segment hydrogen bonds and a closely
packed backbone. Therefore, the stiff α-helical linkers may act
as rigid spacers between protein domains.
Can an expert in the field confirm whether this residue composition is the most suitable? What would be the optimal length?
my fusion protein with 3 linker(GGGGSGGGGSGGGGS)
MCVCRSCRTMSLQKTVEKLFDGGGGSGGGGSGGGGSAELKSALQSCSAEPLDDDHVKAFL
DKGGGGSGGGGSGGGGSKDEPKAHMGQVVKKRWGELRDFFRNDPLGQRLVALGNDLTAIC
QKLQLKIREVLKKYVKNLVEEKDDDSKGGGGSGGGGSGGGGSKDEPKAHMGQVVKKRWGE
LRDFFRNDPLGQRLVALGNDLTAICQKLQLKIREVLKKYVKNLVEEKDDDSK
Thank you for your help.
(Gly4Ser)n linkers actually have very low antigenicity, it is very widely used to connect antibody VL and VH domains in antibody single chain fragments and derived constructs intended for clinical application. It may score high in epitope predictions bases on predicting solvent exposure of subsequences of globular proteins, but it does not contain any hydrophobic side chains needed to provide binding affinity. The optimal linker length depends on the intended application. (Gly4Ser)3, spanning ca 39Å in beta-strand conformation is optimal to bridge a gap of 30-35Å, for other applications smaller or longer linkers may be better, it really depends on the geometric constraints of your system. An alpha-helikal linker with the same number of amino acids will have an end-to-end distance of about 22Å.
Hi, check this out (http://www.sciencedirect.com/science/article/pii/S0169409X12003006)
Indeed, the helical content of the linker you'll design can be estimated using AGADIR (http://agadir.crg.es/). Besides the helicity of your linker, check the hydrophobicity to avoid solubility problems. Hope this help!
there is my domains that i want to link them. I have doubts about the type of linker to use. i want to know weather i choose flexible linkers including Gly-Gly-Gly-Gly-Ser , GGGGGG , or every linker with Gly, or a(EAAAK)3 as Rigid linker on this protein ?
(MCVCRSCRTMSLQKTVEKLFD)
(AELKSALQSCSAEPLDDDHVKAFLDK)
(KDEPKAHMGQVVKKRWGELRDFFRNDPLGQRLVALGNDLTAICQKLQLKIREVLKKYVKNLVEEKDDDSK)
(KDEPKAHMGQVVKKRWGELRDFFRNDPLGQRLVALGNDLTAICQKLQLKIREVLKKYVKNLVEEKDDDSK)
what's your linker suggestion for recombinant short peptide production ?
One method of predicting antigenicity (e.g. to decide what peptides to use as an immunogen to raise antibodies what will cross-react with the full-length folded protein) is to predict which parts of a protein are solvent accessible in the folded structure, therefore "visible" to antibodies.
By this score, Gly-Ser linker ought to be good antigens, they are designed to be solvent accessible. However, they completely lack the hydrophobic surface area that would be needed to get a reasonable affinity for binding to the antibody.
In real life, experimentally, you do not get antibodies that bind to the Gly-Ser linker, although you may get antibodies whose epitope comprises both part of the linker and the linked domain
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