Supramolecular surface immobilization of knottin derivatives for dynamic display of high affinity binders

S. Sankaran, Mark Vincent de Ruiter, Jeroen Johannes Lambertus Maria Cornelissen, Pascal Jonkheijm

Research output: Contribution to journalArticleAcademicpeer-review

14 Citations (Scopus)

Abstract

Knottins are known as a robust and versatile class of miniprotein scaffolds for the presentation of high-affinity binding peptides; however, to date their application in biomaterials, biological coatings, and surface applications have not been explored. We have developed a strategy to recombinantly synthesize a β-trypsin inhibitory knottin with supramolecular guest tags that enable it to adhere to self-assembled monolayers of the supramolecular host cucurbit[8]uril (CB[8]). We have described a strategy to easily express knottins in E. coli by conjugating them to a fluorescent protein after which they are cleaved and purified. Knottin constructs that varied in the number and position of the supramolecular tag at either the N- or C-termini or at both ends have been verified for their trypsin inhibitory function and CB[8]-binding properties in solution and on surfaces. All of the knottin constructs showed strong inhibition of trypsin with inhibition constants between 10 and 30 nM. Using microscale thermophoresis, we determined that the supramolecular guest tags on the knottins bind CB[8] with a Kd of ∼6 μM in solution. At the surface, strong divalent binding has been determined with a Kd of 0.75 μM in the case of the knottin with two supramolecular guest tags, whereas only weak monovalent binding occurred when only one guest tag was present. We also show successful supramolecular surface immobilization of the knottin using CB[8] and prove that they can be used to immobilize β-trypsin at the surface.
Original languageEnglish
Pages (from-to)1972-1980
Number of pages8
JournalBioconjugate chemistry
Volume26
Issue number9
DOIs
Publication statusPublished - 2015

Keywords

  • METIS-314447
  • IR-99556

Fingerprint Dive into the research topics of 'Supramolecular surface immobilization of knottin derivatives for dynamic display of high affinity binders'. Together they form a unique fingerprint.

  • Cite this