Results and Discussion
Chromatographic examination of protein construct integrity as a
function of storage. Figures 1A, and 1B, respectively, show the
mutually-normalized analytical chromatographic gel filtration elution
profiles of the five Coh2-linker-BSX fusion protein constructs. Profiles
shown in Figure 1A were collected immediately after purification, while
profiles shown in Figure 1B were collected after storage at 4 °C for 40
days. In Figure 1A, all constructs are seen to elute as monomers
(between 9.93 and 10.24 ml), with minor differences in elution volume
attributable to known differences in the size(s) of constituent linkers.
Briefly, the constructs roughly eluted in the order of reducing (known)
linker length, and reducing (anticipated) hydrodynamic volume
[Nat-full (9.93 ml; 42 residues), Nat-Quarter (10.15 ml;
9 residues), Nat-half (10.18 ml; 21 residues), Rigid(10.20 ml; 15 residues), and Flexible (10.24 ml; 10 residues)],
with only Nat-quarter behaving anomalously. Figure 1B shows that,
after 40 days of storage at 4 °C, the single elution peaks of the
constructs (between 9.93 and 10.24 ml), degenerated into two new peaks
of significance, at ~11.5 ml, and ~13.5
ml, with four of the five constructs (incorporating Nat-Quarter,
Nat-half, Nat-full and Rigid linkers); two additional minor
peaks at ~19 and ~20 ml with two of the
five constructs (incorporating Nat-full and Nat-half ); but
no peaks whatsoever at ~13.5, ~19 or 20
ml, and only an insignificant peak at ~11.5 ml, with the
fifth construct (incorporating Flexible ). This demonstrates that
four constructs underwent significant degradation, whereas the construct
incorporating Flexible did not undergo degradation. The species
eluting at ~11.5 ml, and ~13.5 ml,
respectively, were confirmed to be BSX, and Coh2, through control
experiments. Since BSX and Coh2 are thermostable, they resist
proteolysis. Therefore, trace amounts of proteases in the proteins’
environment attack only the linker backbones in four of the five
Coh2-linker-BSX constructs, to physically separate BSX and Coh2 and
cause them to elute independently.