Multifunctional, High Molecular Weight, Post-Translationally Modified Proteins through Oxidative Cysteine Coupling and Tyrosine Modification.

Glycoproteins and their mimics are challenging to produce via chemical or biological methods because of their long protein backbones and large number of polysaccharide side chains that form a densely grafted protein-polysaccharide brush architecture. Herein, we demonstrate a new approach to protein bioconjugate synthesis that can approach the molar mass and functionalization densities of natural glycoproteins such as mucins and aggrecans. In this method, a tyrosine-enriched protein sequence is engineered and synthesized in E. coli, and sugars or other functional moieties can be efficiently and polyvalently grafted to the backbone through tyrosine modification chemistry. Cysteine residues on the chain ends are used for oxidative chain polymerization into high molar mass chains larger than can be easily expressed in the host. The effects of tyrosine-enrichment and cysteine-incorporation on the physical and expression properties on a model protein are explored. Elastin-like peptides (ELPs) are chosen because of their high expression yields, repetitive sequence, substitutable amino acids, and well-studied physical properties. The sequence modifications to mimic glycoproteins are shown to affect the maximum length of expressible sequence but not yield. The tyrosine modification chemistry is shown to functionalize up to 73% of all tyrosines on the peptide, and the scope of functional groups that can be mass conjugated to proteins is expanded through multistep conjugation strategies involving copper(I)-catalyzed alkyne-azide cycloaddition showing up to 97% alkyne functionalization. All of the functionalization chemistries preserve the ability to polymerize the backbone.