Date Published: May 24, 2019
Publisher: Public Library of Science
Author(s): Shikha Singh, Kanchan Gupta, Shagun Shukla, Srinivasa-Gopalan Sampathkumar, Rajendra P. Roy, Joseph J. Barchi.
Multivalent proteins or protein dendrimers are useful for clinical and biotechnological applications. However, assembly of chemically defined protein dendrimers is a challenging endeavor. In the past, majority of protein dendrimers have been developed on branched lysine scaffolds and are usually limited to a valency of two to four. The naturally occurring cyclodextrin (CD) scaffold composed of 6–8 glucose units offers the possibility of expanding the valency. Here we have adapted a chemoenzymatic-click strategy for displaying heptavalent peptides and large proteins on the β-cyclodextrin (β-CD) scaffold. We demonstrate that recombinant proteins (engineered with a LPXTG pentapeptide motif at the carboxy terminus), labeled with an alkyne moiety by sortase-mediated ligation, can be easily clicked on to the azide-derivatized β-cyclodextrin through the Huisgen cycloaddition reaction yielding a well-defined heptavalent display of proteins.
Cyclodextrins (CD) are natural cyclic oligomers composed of six (α), seven (β) or eight (γ) D-glucose units linked by α- 1, 4- glycosidic bonds . Cyclodextrins have a torus architecture with a hydrophobic cavity that allows inclusion of spatially compatible molecules [2–4]. The noncovalent inclusion complexes of cyclodextrin have attracted a wide variety of applications for stabilization of drugs , solubilization of peptides and proteins, protein folding  etc. Besides, CD scaffold also comprises one of the most easily accessible scaffolds for multivalent display of ligands. The hydroxyl groups on CD surface are endowed with differential reactivity and are amenable to suitable modifications with azide, alkynes, esters, sugars and other functionalities for further elaboration with a variety of macromolecules derivatized with compatible orthogonal groups [7–11].
Fmoc-propargylglycine (Fmoc-D-Pra-OH) was purchased from Anaspec, USA. Fmoc-Gly and Wang resin was procured from Novabiochem, USA. 1-Hydroxybenzotriazole (HOBt) was purchased from GL Biochem, China. Oligonucleotide Primers were custom synthesized from Sigma-Aldrich, USA. PCR reagents, Taq Polymerase (Platinum HiFi) was obtained from Invitrogen, USA, Plasmid Miniprep kits, Gel extraction buffers, PCR purification kits, Ni-NTA beads, were obtained from Qaigen,USA. pET23b plasmid, T7 promoter /terminator primer was supplied by Novagen Inc., USA. All other chemicals and solvents used in the study were obtained from Sigma-Aldrich, USA.
Assembly of chemically well-defined protein dendrimers by purely chemical methods is limited by the sequence length and associated purification complexities. These limitations have been partly overcome by advances in peptide fragment condensation and click chemistry approaches. However, the lack of a general route for assembly of a dendrimer composed of large proteins remains a formidable problem. This is further underscored by the description of limited examples of defined protein dendrimers in literature. Some examples include a trivalent antigen binding construct built on a trialdehyde scaffold using an aminoxy derivatized Fab fragment , divalent and tetravalent dendrimers of GFP or collagen binding protein CNA35 by NCL using branched peptides terminating with N-terminus Cys residues and respective thioester derivatized proteins . A combinatorial approach of native chemical ligation (NCL) and CuAAC was also explored with a tripropargylamine scaffold for the assembly of trivalent protein G dendrimers . Recently, we exploited the peptide ligation propensity of transpeptidase sortase for installing orthogonal groups compatible with click chemistry for synthesis of chemically defined protein dendrimers . A similar two-step approach using a combination of sortase-mediated labelling and click chemistry was earlier employed for site-specific modification of recombinant antibodies .