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.