Three tandem GGGGS flexible linker

Flexible glycine-serine linker encoding SGGGGSGGGGSGGGGS, a serine followed by three tandem GGGGS repeats. Joins two protein domains through a flexible, hydrophilic spacer that does not interfere with their folding. (GGGGS)n is the most widely used flexible linker, and the repeat number n sets the separation between domains.

Length: 48 bp

Subtype: flexible_linker

Origin: Synthetic glycine-serine repeat

Characteristics

Encodes SGGGGSGGGGSGGGGS (16 amino acids, 48 nucleotides): a serine plus three GGGGS repeats. Small Gly residues provide flexibility and mobility between the joined domains, and Ser forms hydrogen bonds with water, which keeps the linker stable in aqueous solution and reduces unfavorable interaction with the fused proteins. The 15-residue (GGGGS)3 linker of the original anti-digoxin scFv has no leading serine. Its calculated length is about 5.7 nm, compared with 3.5 nm between the VH C terminus and VL N terminus in their natural orientation. A flexible linker acts as a passive spacer.

Applications: Joins the VH and VL domains of single-chain Fv (scFv) antibody fragments. In the anti-digoxin 26-10 scFv made in E. coli, the 15-residue (GGGGS)3 linker gave a refolded scFv with a digoxin association constant of 3.2 x 10^7 M-1, about 6-fold below the parent Fab. Between G-CSF and transferrin (Tf), (GGGGS)3 improved in vitro G-CSF bioactivity over the unlinked fusion, which retained less than 10% of parent activity.

Limitations: A flexible linker can fail to separate domains adequately and gives no fixed distance. A Tf-G-CSF fusion with (GGGGS)3 was almost undetectable in expression, while a helical (H4)2 linker gave high expression of the same fusion. The optimal repeat number depends on the fusion and must be tuned.

Sequence

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References

  1. Huston et al. (1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA - Huston 1988 Single-Chain Fv Linker
  2. Chen et al. (2012). Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev - Chen 2013 Fusion Linkers