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 .