Choosing an effective protein bioconjugation strategy (1)
A ‘bioconjugation flowchart’ provides a simple guide that can be used to select an appro- priate technique in many instances. Lysine modification chemistry is often the best choice. A large number of N-hydroxysuccinimide (NHS) esters and isothiocyanates are available, and lysine residues can also be modified through reductive alkylation with aldehydes and reducing agent. One must probe the inherent reaction landscape of the particular protein by targeting its cysteines, tyrosines, tryptophans and N-terminal groups, often by trial and error. Unique cysteines can be alkylated using maleimide reagents, iodoacetamides, vinyl sul- fones and acrylamides, and they can be linked to new functionality through disulfide formation with activated thiol reagents. As with lysine-modification reagents, there are many com- mercially available maleimides and iodoacetamides that are ready for use. The N terminus can also be targeted through serine oxidation or transamination, and specific N-terminal amino acids can react chemoselectively with aldehydes through oxazolidine (with serine) and thiazolidine (with cysteine) formation or through Pictet-Spengler reactions with tryptophan.
The genome-free protein capsids have been explored by our laboratory as targeted carri- ers that can house drug molecules and imaging agents and deliver them to cancer targets. Figure 2b arrives at the installation of a cysteine residue on the interior surface to allow modification with maleimide-bearing cargo molecules. a new highly reactive cysteine could be introduced in position 87, providing 180 alkylation sites inside the shell. A native tyrosine residue (Tyr85) can also be modified selectively using elec- tron-deficient diazonium salts, providing a useful alternative for interior labeling when maleimide reagents are unavailable or inconvenient.
There is the use of the E. coli enzyme lipoic acid ligase (LplA)76, which catalyzes the ATP-dependent attachment of lipoic acid to one of three native protein targets involved in oxidative metabolism. Similar to LplA, biotin ligase (BirA) modifies a short acceptor peptide with biotin.
