Bioproduction of highly charged designer peptide surfactants via a chemically cleavable coiled-coil heteroconcatemer

Fletcher, Nicholas L., Paquet, Nicolas, Dickinson, Ellyce L. and Dexter, Annette F. (2015) Bioproduction of highly charged designer peptide surfactants via a chemically cleavable coiled-coil heteroconcatemer. Biotechnology and Bioengineering, 112 2: 242-251. doi:10.1002/bit.25446

Author Fletcher, Nicholas L.
Paquet, Nicolas
Dickinson, Ellyce L.
Dexter, Annette F.
Title Bioproduction of highly charged designer peptide surfactants via a chemically cleavable coiled-coil heteroconcatemer
Journal name Biotechnology and Bioengineering   Check publisher's open access policy
ISSN 1097-0290
Publication date 2015-02
Year available 2014
Sub-type Article (original research)
DOI 10.1002/bit.25446
Open Access Status
Volume 112
Issue 2
Start page 242
End page 251
Total pages 10
Place of publication Hoboken NJ, United States
Publisher John Wiley & Sons
Collection year 2015
Language eng
Formatted abstract
Designer peptides have recently attracted attention as self-assembling fibrils, hydrogelators and green surfactants with the potential for sustainable bioproduction. Carboxylate-rich peptides in particular have shown potential as salt-resistant emulsifiers; however the expression of highly charged peptides of this kind remains a challenge. To achieve expression of a strongly anionic helical surfactant peptide, we paired the peptide with a cationic helical partner in a coiled-coil miniprotein and optimized the polypeptide sequence for net charge, hydropathy and predicted protease resistance (via the Guruprasad instability index). Our design permitted expression of a soluble concatemer that accumulates to high levels (22% of total protein) in E. coli. The concatemer showed high stability to heat and proteases, allowing isolation by simple heat and pH precipitation steps that yield concatemer at 133 mg per gram of dry cell weight and >99% purity. Aspartate-proline sites were included in the concatemer to allow cleavage with heat and acid to give monomeric peptides. We characterized the acid cleavage pathway of the concatemer by coupled liquid chromatography-mass spectrometry and modeled the kinetic pathways involved. The outcome represents the first detailed kinetic characterization of protein cleavage at aspartate-proline sites, and reveals unexpected cleavage preferences, such as favored cleavage at the C-termini of peptide helices. Chemical denaturation of the concatemer showed an extremely high thermodynamic stability of 38.9 kcal mol−1, with cleavage decreasing the stability of the coiled coil to 32.8 kcal mol−1. We determined an interfacial pressure of 29 mN m−1 for both intact and cleaved concatemer at the air-water interface, although adsorption was slightly more rapid for the cleaved peptides. The cleaved peptides could be used to prepare heat-stable emulsions with droplet sizes in the nanometer range.
Keyword Concatemer
Charge pairing
Alpha helix
Instability index
De novo design, acid cleavage
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Published online ahead of print 10 Oct 2014

Document type: Journal Article
Sub-type: Article (original research)
Collections: School of Chemical Engineering Publications
Official 2015 Collection
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Citation counts: TR Web of Science Citation Count  Cited 1 times in Thomson Reuters Web of Science Article | Citations
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