Figure 1.
Schematic illustration of sortase A mediated reaction fusing two proteins.
Sortase A fuses an LPXTG recognition motif to an N-terminal GGG motif, regenerating a native amide bond.
Figure 2.
Structures of N and C-terminal fusion partners denoting site and sequence of sortase A recognition motifs of each domain.
Figure 3.
Sortassembled products and functional testing.
Nonreduced gel (A) and reduced Western blot (B) demonstrating the shift in molecular weight of an IgG light chain-LPETGXn following fusion with 5 different triglycine partners. Fusion protein products are marked with an asterisk. Light Chain (LC) was visualized. C) Gel demonstrating shift of A33 antigen-LPETGXn following fusion in reactions (rxn) with 5 different triglycine partners. Fusion products are marked with an asterisk. D,E) Functional tests: fluorescence histograms of A33 IgG fused to GFP (D), and CEA IgG fused to A33 antigen (E). Blue trace represents reaction mix, remaining traces represent bare beads (red), reaction mix without sortase enzyme (green), and reaction mix with competitor IgG (yellow).
Figure 4.
Optimizing reaction conditions.
A) Fusion protein yield in a reaction time course demonstrating maximal yield between 1 and 4 hours, and reduced yield at prolonged reaction times. B) Product yield at varying pH demonstrating good yield at pH values between 7 and 9. C) Yield at different temperatures, demonstrating improved yield at temperatures slightly above 37°C.
Figure 5.
Optimizing concentrations of reactants.
A) Fusion protein yield at differing ratios of reactants indicating that high concentrations of both reactants ought to be used. B) Gel of fusion product demonstrating that excess of either reactant pushes reaction toward completion.
Figure 6.
Optimizing enzyme concentrations.
A) Fusion product yield in reactions with equimolar reactants, while varying the ratio of sortase∶total reactants. B) Fusion product yield with varying amounts of sortase enzyme in reactions with a 10-fold excess of triglycine reactant over LPETG reactant.
Table 1.
Optimized conditions for sortassembly.
Figure 7.
Product quantification and purification.
A) Western blot showing amount of unreacted LPETGXn substrate before (left) and after (right) reaction, demonstrating an 85% yield when bands are quantified. B) Example purification demonstrating separation of unreacted A33-LPETG-His6 and sortase-His6 enzyme from reaction mixture by nickel-based affinity chromatography.