Figure 1. Chromatographic examination of protein construct integrity as a function of storage, throughmonitoring of gel filtration chromatographic elution profiles. Panel A. Gel filtration chromatography profiles of all constructs on the day of purification.Panel B. Gel filtration chromatography profiles of all constructs after 40 days of storage in a refrigerator.
Gel filtration chromatographic results do not, however, address the question of whether there have been any additional intra-domain proteolytic cuts within the backbones of BSX and Coh2. To examine whether there are any such cuts, we performed SDS-PAGE experiments (under denaturing conditions), to investigate whether any sections of Coh2, or BSX, had become covalently separated from other sections, to remain remain non-covalently associated during chromatography.
Electrophoretic examination of protein construct integrity as a function of storage. Figure 2A presents SDS-PAGE profiles of constructs collected immediately after purification, demonstrating the absence of degradation. On a 12 % SDS-PAGE, each construct displayed a mobility attributable to the size of the extended (i.e., SDS-bound, and SDS- and heat-unfolded) polypeptide, with the construct incorporatingNat-full showing the least mobility due to its possession of the longest linker sequence, and constructs incorporating Nat-halfand Rigid displaying mobilities intermediate to those of constructs incorporating Nat-full, on the one hand, andFlexible and Nat-Quarter , on the other hand. Constructs incorporating Flexible and Nat-Quarter display the highest mobility because they have the smallest linkers. No construct displayed signs of anomalous mobility, or significant degradation. Figure 2B presents the SDS-PAGE profiles collected after 15 days of storage at 4 °C, by which time (despite the anticipated lower activity of proteolytic enzymes naturally present in the environment, at low temperatures; unless derived from psychrophile organisms in refrigerators), sufficient degradation was observed to have already occurred in four constructs. Figure 2B shows that Coh2, and BSX, being well-folded domains with folding equilibria keeping the bulk of their populations folded at any time, largely remain intact and undergo no visible degradation, either while they exist in fusion, or after their separation through proteolytic scission of the linker, as is evident from the fact that there is no lowering in intensity of bands corresponding to intact Coh2-linker-BSX that are not compensated for by the intensities of bands corresponding to free BSX, or Coh2. No visible bands suggest degradation of BSX and Coh2 and, in each case, in Figure 2B, degradation produces three distinct species: (i) the residual (un-degraded) Coh2-linker-BSX [size > 60 kDa] population; (ii) a population corresponding to BSX [size ~45 kDa], and (iii) a population corresponding to Coh2, with the linker peptide still present at the C-terminus [size > 20 kDa). Figure 2B thus establishes that linkers undergo proteolysis at their BSX-adjoining ends, in constructs incorporating Nat-Quarter, Nat-half, Nat-fulland Rigid .