PPubMed7 Oct 2019Cited 15×

Fabrication of rigidity and space variable protein oligomers with two peptide linkers † †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc04158c

Hyeokjune Choi, Hyoin Park, Kabi Son, Ho Min Kim, Yongwon Jung

Abstract

A new two peptide-linked protein assembly strategy was developed for diverse linker length and rigidity variation on protein oligomers.

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A two‑peptide linker strategy creates GFP oligomers with tunable length and rigidity, enabling multivalent protein scaffolds up to decamer size.

What’s new

i.Developed a two‑peptide linked protein assembly method allowing systematic variation of linker length and rigidity
ii.Fabricated GFP oligomers with the longest reported peptide linkers (up to 76 aa rigid and 38 aa flexible)
iii.Demonstrated multivalent display of diverse functional proteins on oligomers ranging from monomer to decamer
iv.Showed that linker spacing influences multivalent binding strength using surface plasmon resonance
v.Provided extensive biophysical characterization (TEM, DLS, CD, DSC, SEC) of the assembled oligomers

How they did it

Split superfolder GFP fragments (GFP 1‑9, 10, 11) were genetically fused via two designed peptide linkers and expressed in E. coli. The assembled oligomers were purified by Ni‑affinity chromatography and gel electro‑elution, then analyzed with native PAGE, SEC, TEM, DLS, CD, DSC and SPR to assess structure and function.

What they found

Over 100 mg of GFP oligomers obtained from 1 L culture; 2‑4 mg of trimer/tetramer and 1‑2 mg of 5‑10 mer per litre
~43 % of 10aa/10aa building blocks polymerized into ≥3‑mer oligomers
Longest two‑peptide linkers combined a 38‑aa flexible and a 76‑aa rigid peptide, the longest reported for protein subunit linkage
Oligomers remained stable at >0.3 mg mL⁻¹ for ≥6 months and tolerated heating up to ~60 °C (first melting) and ~67 °C (full denaturation)
SPR revealed enhanced binding stability for spaced (H4)3/38aa oligomers compared with shorter 15aa/15aa linkers

Caveats

✕Direct structural confirmation of highly spaced, flexible assemblies is difficult, as TEM images are heterogeneous
✕Overall production yields and the multi‑step purification process are still low and complex
✕Very large oligomers (e.g., (H4)4/50aa) could not be well separated on native gels
✕Further methods are needed to verify structures of flexible supramolecular protein architectures

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Fabrication of rigidity and space variable protein oligomers with two peptide linkers † †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc04158c

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