Coiled Coil Peptides as Universal Linkers for the Attachment of Recombinant Proteins to Polymer Therapeutics

Abstract

We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.

Location of the Bacteriophage P22 Coat Protein C-Terminus Provides Opportunities for the Design of Capsid-Based Materials

“…Amino acid sequences corresponding to either a positive (CP-K-coil, +TR(VAALKEK) 3 ) or negatively charged (CP-E-coil, +TS(VAALEKE) 3 ) alpha helix of a three heptad heterodimeric coiled coil motif were appended to the C-terminus of the coat protein. Peptide sequences, VAALKEK 3 and VAALEKE 3 , have been previously described to form a anti-parallel coiled coil heterodimer upon association 25 . The genetic amenability of the C-terminal location is reflected in the addition of these 23 residues without noticable alterations to the assembly of the P22 procapsid.…”

Multimodality Imaging of Coiled‐Coil Mediated Self‐Assembly in a “Drug‐Free” Therapeutic System

“…developed a new strategy of using recombinant DNA technology to incorporate a coiled-coil peptide into a single chain (scFv) fragment. [17,18] This fusion protein approach could produce a high amount of well-defined scFv-peptide conjugates directly. More importantly, the coiled-coil peptide will have a minimal effect on the structure and biological activity of the scFv fragment.…”

“…[3] Since nature has provided us with such perfect motif for molecular recognition and assembly, much attention has been attracted to the applications of self-assembling coiled-coil peptides for engineering functional materials, such as multimeric antibodies, epitope display, affinity purification, tissue engineering, regenerative medicine, biosensor, nanomaterials assembly, and drug delivery. [4-18] In our group, two complementary coiled-coil peptide sequences (CCE and CCK) were previously designed and successfully assembled hybrid graft copolymers into hydrogels. [19,20] Recently, this coiled-coil pair was employed in a therapeutic system to specifically trigger apoptosis in cancer cells.…”

Antibody-pHPMA functionalised fluorescent silica nanoparticles for colorectal carcinoma targeting

“…[18][19][20] Other studies conrmed that pHPMA can create a stealth hydrophilic layer or even enhance the blood circulation in the case of DNA complexes, 21 selfassembled vesicles, 22 polymeric particles 23 or viral vectors.…”