Increase in cysteine-mediated multimerization under attractive protein-protein interactions.

Area of Science:

• Biochemistry
• Protein Chemistry
• Enzymology

Background:

• The CASPON enzyme is valuable for fusion protein processing due to its ability to create an authentic N-terminus.
• High cysteine content in CASPON can lead to aggregation via disulfide bonds, reducing enzymatic activity and impacting quality.

Purpose of the Study:

• To investigate the multimerization states of the CASPON enzyme.
• To analyze the impact of co-solutes on CASPON enzyme multimerization and activity.
• To understand the role of cysteine in CASPON enzyme aggregation and activity.

Main Methods:

• Isolation of CASPON enzyme multimerization states using preparative size exclusion chromatography.
• Analysis of multimerization propensity and enzymatic activity.
• Study of co-solute effects on multimerization in solution and adsorbed states.
• Measurement of protein-protein interactions using self-interaction chromatography.

Main Results:

• The dimer form of CASPON enzyme exhibited the highest stability and 50% greater enzymatic activity than the tetramer.
• Cysteine-mediated pathways were identified as the primary drivers of CASPON enzyme multimerization, evidenced by DTT reduction.
• Ammonium sulfate promoted attractive protein-protein interactions, correlating with increased multimerization via the cysteine pathway.
• Multimerization was also observed under attractive conditions on chromatographic stationary phases.

Conclusions:

• The dimer is the optimal and most active form of the CASPON enzyme.
• Cysteine-mediated disulfide bond formation significantly influences CASPON enzyme aggregation and activity.
• Protein purification strategies should minimize residence time to prevent aggregation and conformational changes, especially on chromatographic surfaces.