Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 141, Issue 9, Pages 3875-3884Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.8b10734
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Funding
- Army Research Office [W911NF-15-1-0144, W911NF-16-1-0169]
- BASF CARA program
- Chemical Biology Graduate Program at UC Berkeley [NIH T32-GM066698]
- DoD, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEG) Fellowship [32 CFR 168a]
- NIH Shared Instrumentation Grant [S10OD010786]
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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 (VLPs) composed of 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.
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