Association of Zinc Supplementation with Recombinant Protein Recovery and Crystallization

Reproducible production of stable recombinant proteins is essential for biochemical and structural studies, but conventional optimization may be insufficient when constrained by protein-specific requirements.In this study, we investigated batch-to-batch variation in the recombinant production of a putative Zn 2+ -dependent metalloprotease from Cutibacterium acnes.Protein expression and purification yields varied despite identical procedures, and conventional optimization failed to restore consistency.AlphaFold2 modeling and comparison with a Zn 2+ -bound homolog suggested a conserved Zn 2+ -binding site.Protein production was therefore compared between cultures grown with and without Zn 2+ supplementation.The Zn 2+ -supplemented batch exhibited a stronger target-protein band on SDS-PAGE and a larger A 280 peak on size-exclusion chromatography, indicating greater protein recovery.Under identical crystallization conditions, the unsupplemented protein predominantly formed microcrystals of approximately 50 μm, whereas the Zn 2+ -supplemented protein formed larger crystals of approximately 300-500 μm.These findings associate Zn 2+ supplementation with increased protein recovery and larger crystal formation, highlighting the importance of considering protein-specific cofactor requirements during recombinant protein production.