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<p>Site-specific protein modification is a widely-used
strategy to attach drugs, imaging agents, or other useful small molecules to
protein carriers. N-terminal modification is particularly useful as a
high-yielding, site-selective modification strategy that can be compatible with
a wide array of proteins. However, this modification strategy is incompatible
with proteins with buried or sterically-hindered N termini, such as virus-like
particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs
with improved compatibility with these techniques, we generated a targeted
library based on the MS2-derived protein cage with N-terminal proline residues
followed by three variable positions. We subjected the library to assembly,
heat, and chemical selections, and we identified variants that were modified in
high yield with no reduction in thermostability. Positive charge adjacent to
the native N terminus is surprisingly beneficial for successful extension, and
over 50% of the highest performing variants contained positive charge at this
position. Taken together, these studies described nonintuitive design rules
governing N-terminal extensions and identified successful extensions with high
modification potential.</p>
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