This paper describes an approach for synthesizing megamolecules through a step-wise joining of a fusion protein prepared from cutinase (blue) and HaloTag (green) and of a bifunctional linker terminated in irreversible inhibitors for the proteins. A) The fusion protein (HC) can be blocked at the cutinase or HaloTag domain by reaction with a phosphonate or hexylchloride group, respectively. B) A dimeric structure can be assembled by allowing the fusion protein to react with the linker followed by another equivalent of HC protein. C) Repetition of this cycle gives oligomeric forms of the HC protein. The bifunctional linker is terminated in p-nitrophenylphosphonate (yellow) and an alkylchloride (purple) group (bottom). The proteins in panels a and b are shown to scale and those in panel c are shown in perspective with respect to the plane of the page.

Kinetic analysis of the reactions of HaloTag-Cutinase (HC) fusions and linker molecules. A) The HC fusion protein was treated with a ligand to block either the cutinase (left) or HaloTag (right) domain and then treated with the linker for specified times before treating with an excess of an inhibitor-fluorophore conjugate to label the remaining unreacted protein. The yields for each reaction were determined by monitoring the loss of the starting protein by gel electrophoresis and the data were fit to determine the first order rate constant for the reaction. B) This experiment was repeated to measure the rate constant for the coupling of two fusion proteins, as described in the text.

The molecular weights of the oligomers that were prepared as outlined in were determined using (A) SDS-PAGE analysis and (B) MALDI mass spectrometry. The inset is a zoom of the MALDI spectrum for the pentameric product around the expected m/z value.

Hydrodynamic characterization of oligomeric reaction products. Size exclusion chromatography traces are shown for each species. Hydrodynamic radii were determined using globular protein calibrants.

We measured the rate constants for reaction of the HaloTag and cutinase domains of the fusion protein with the bifunctional linker. In one example, we first blocked the cutinase domain of the HC fusion protein with 4-nitrophenyl-(6-carboxyhexyl)-1-phosphonate (4-NPCP) and then allowed the HaloTag domain (at 1 μM) to react with the linker (20 μM) in separate reactions for times ranging from 0 to 600 seconds. We stopped the reactions by adding a large excess of a chloroalkyl-AlexaFluor 488 conjugate to label the unreacted HaloTag with a fluorophore. The reaction mixtures were then resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of fluorescently-labeled protein was measured on a gel scanner and used to determine the yield for each reaction (). Reaction of the protein (E) with the linker (L) proceeds by way of an enzyme-substrate complex (). Because the rate constant for the chemical step (k₂) is slow relative to that for dissociation of the E-L complex (k₋₁), the rate law for the reaction is first order with respect to both the enzyme and the linker (). Further, because the concentration of linker was 20-fold greater than that of the protein, we fit the kinetic data to a pseudo-first order model to obtain a second order rate constant, k_eff, of 715 ± 79 M⁻¹s⁻¹ for the reaction of the HaloTag domain with the linker. We repeated this experiment with HC that was first treated with an alkylchloride inhibitor to block the HaloTag domain and found that cutinase reacts with the bifunctional linker with a second order rate constant of 30 ± 5 M⁻¹s⁻¹.

We measured the rate constants for reaction of the HaloTag and cutinase domains of the fusion protein with the bifunctional linker. In one example, we first blocked the cutinase domain of the HC fusion protein with 4-nitrophenyl-(6-carboxyhexyl)-1-phosphonate (4-NPCP) and then allowed the HaloTag domain (at 1 μM) to react with the linker (20 μM) in separate reactions for times ranging from 0 to 600 seconds. We stopped the reactions by adding a large excess of a chloroalkyl-AlexaFluor 488 conjugate to label the unreacted HaloTag with a fluorophore. The reaction mixtures were then resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of fluorescently-labeled protein was measured on a gel scanner and used to determine the yield for each reaction (). Reaction of the protein (E) with the linker (L) proceeds by way of an enzyme-substrate complex (). Because the rate constant for the chemical step (k₂) is slow relative to that for dissociation of the E-L complex (k₋₁), the rate law for the reaction is first order with respect to both the enzyme and the linker (). Further, because the concentration of linker was 20-fold greater than that of the protein, we fit the kinetic data to a pseudo-first order model to obtain a second order rate constant, k_eff, of 715 ± 79 M⁻¹s⁻¹ for the reaction of the HaloTag domain with the linker. We repeated this experiment with HC that was first treated with an alkylchloride inhibitor to block the HaloTag domain and found that cutinase reacts with the bifunctional linker with a second order rate constant of 30 ± 5 M⁻¹s⁻¹.