Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

Abstract

Site-Specific Modification. ChemRxiv. Preprint. 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. File list (3) download file view on ChemRxiv ChemRxiv Submission Brauer Hartman.pdf (2.67 MiB) download file view on ChemRxiv Supplementary Information Brauer Hartman.pdf (2.78 MiB) download file view on ChemRxiv Supplementary Data-Primers.xlsx (16.69 KiB)

Direct observation of ion emission from charged aqueous nanodrops: effects on gaseous macromolecular charging

“…Bacteriophage MS2 virus-like particles were produced as previously described. 46 In brief, DH10B E. coli containing a pBAD MS2 expression vector (chloramphenicol resistant) was grown to OD0.6 in 2xYT media at 37 C. Expression was induced via addition of arabinose to 0.1% (w/v) and cultures were incubated for 18 h at 37 C. Cells were then harvested via centrifugation and lysed by sonication. The collected lysate was puried through two rounds of ammonium sulfate precipitation at 50% saturation followed by polishing on anÄKTA start FPLC system using a prepacked Sephacryl S-500 HR column.…”

Hierarchical Self-Assembly of Proteins Through Rationally Designed Supramolecular Interfaces

“…If rationally arranging chromophores on the similar structures, the protein nanomaterials could be designed to construct artificial light harvesting systems (Sun et al, 2016a). Besides, other kinds of functional materials have been developed, such as viral capsid grafting for targeted imaging/drug delivery (Brauer et al, 2019) and nanoreactors (Jordan et al, 2016), artificial metalloenzymes construction on protein structures (Ueno et al, 2013), artificial GPx nanoenzyme on GST nanowires (Hou et al, 2013), and others.…”

Engineering a Virus-like Particle to Display Peptide Insertions Using an Apparent Fitness Landscape

“…RNA was extracted from the FG loop (NNK)3 library following assembly using previously published protocols. [17][18][19] In summary, TRIzol (Thermo Fisher, catalog no. 15596026) was used to homogenize collected VLP-containing fractions after assembly selections, and an equal volume of chloroform was added.…”

“…Data were trimmed and processed as previously described with minor variations. [17][18][19] Briefly, data were trimmed with Trimmomatic with a four-unit sliding quality window of 20 and a minimum length of 30.…”

“…We applied this technique to explore the mutability and principles of higher order assembly for the MS2 VLP. [17][18][19] Through this work we identified a region known as the "FG loop" to be especially permissive to mutations. This loop is unique in its conformations and placement in the packing of MS2 monomers.…”