Supramolecular Surface Immobilization of Knottin Derivatives for Dynamic Display of High Affinity Binders.
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.
Bioinspired Molecular Engineering Laboratory of the MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente , 7500 AE Enschede, The Netherlands.
Shrikrishnan
S
Sankaran
Mark
M
de Ruiter
Jeroen J L M
JJ
Cornelissen
Bioinspired Molecular Engineering Laboratory of the MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente , 7500 AE Enschede, The Netherlands.
Pascal
P
Jonkheijm
D001952:Bridged-Ring Compounds / Q000737:chemistry; D057771:Cystine-Knot Miniproteins / Q000737:chemistry* / Q000494:pharmacology; D007093:Imidazoles / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry* / Q000494:pharmacology; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D013499:Surface Properties; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000494:pharmacology; D047028:Macrocyclic Compounds; D000099246:Cucurbit(n)urils
D001952:Bridged-Ring Compounds; D057771:Cystine-Knot Miniproteins; D007093:Imidazoles; D055767:Immobilized Proteins; D014361:Trypsin Inhibitors; C507198:cucurbit(8)uril; D047028:Macrocyclic Compounds; D000099246:Cucurbit(n)urils