title: Surface functionalization of virus-like particles by direct conjugation using azide-alkyne click chemistry.
abstract: We present a cell-free protein synthesis (CFPS) platform and a one-step, direct conjugation scheme for producing virus-like particle (VLP) assemblies that display multiple ligands including proteins, nucleic acids, and other molecules. Using a global methionine replacement approach, we produced bacteriophage MS2 and bacteriophage Qβ VLPs with surface-exposed methionine analogues (azidohomoalanine and homopropargylglycine) containing azide and alkyne side chains. CFPS enabled the production of VLPs with yields of ~ 300 μg/mL and with 85% incorporation of methionine analogues without requiring a methionine auxotrophic production host. We then directly conjugated azide- and alkyne-containing proteins (including an antibody fragment and the granulocyte-macrophage colony stimulating factor, or GM-CSF), nucleic acids and poly(ethylene glycol) chains to the VLP surface using Cu(I) catalyzed click chemistry. The GM-CSF protein, after conjugation to VLPs, was shown to partially retain its ability to stimulate the proliferation of cells. Conjugation of GM-CSF to VLPs resulted in a 3-5-fold reduction in its bioactivity. The direct attachment scheme facilitated conjugation of three different ligands to the VLPs in a single step, and enabled control of the relative ratios and surface abundance of the attached species. This platform can be used for the production of novel VLP bioconjugates for use as drug delivery vehicles, diagnostics, and vaccines.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
authors.forename: Kedar G
authors.initials: KG
authors.lastname: Patel
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000480:Alkynes / Q000737:chemistry*; D000818:Animals; D000941:Antigens / Q000276:immunology; D002460:Cell Line; D002474:Cell-Free System / Q000378:metabolism; D057930:Click Chemistry; D018899:CpG Islands; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D007130:Immunoglobulin Idiotypes / Q000276:immunology / Q000378:metabolism; D016393:Lymphoma, B-Cell / Q000276:immunology; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry; D013379:Substrate Specificity; D013499:Surface Properties; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
chemical_list: D000480:Alkynes; D000941:Antigens; D007130:Immunoglobulin Idiotypes; D058425:Vaccines, Virus-Like Particle; C506098:azidohomoalanine; C551041:homopropargylglycine; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; D000409:Alanine; D005998:Glycine