Extended Data Fig. 2: Design and electrophoretic gel visualization of self-assembling, disulfide-stapled cTRP24 nanoparticles.

a, The cTRP2412SS construct, assembled from dimerization of two identical protein subunits each harboring 12 repeats, with the N- and C-terminal repeat of each containing a cysteine residue (described in the main text and in Fig. 2) that enable disulfide stapling. SEC analyses (shown in Fig. 2c) and electrophoretic analyses (right panel) both indicate formation of a dimer that contains a mixture of one or two disulfide staples, both of which behave in solution similarly to a monomeric, single chain 24-repeat cTRP. b, A cTRP246SS construct, assembled from four identical protein subunits each harboring 6 repeats, with the N- and C-terminal repeat of each containing a cysteine residue (described in the main text and in Fig. 2) that enable disulfide stapling. SEC analyses indicate that expression and purification yield a cTRP24 that behaves in solution in a similar manner to a monomeric, single chain 24-repeat cTRP. Electrophoretic analyses of the same construct generated either via cytosolic expression in E. coli (and then oxidized in the presence of air during purification) or via secretion from human HEK cells (oxidized as part of eukaryotic disulfide bond formation mechanism during secretion) indicate that disulfides are formed in both cases. However, expression from bacteria generates a mixture of species (harboring 2, 3 or 4 disulfides), whereas expression and secretion from human cells generated a more homogeneous population of species primarily consisting of a full complement of disulfide bonds.