Efficient periplasmic expression of active lysyl endopeptidase and optimizing the purification methods

Area of Science:

• Biotechnology
• Protein Expression
• Enzymology

Background:

• Recombinant production of lysyl endopeptidase (Lys-C) is challenging due to its complex structure.
• Lys-C is a crucial enzyme in proteomics for protein digestion.

Purpose of the Study:

• To optimize periplasmic expression of Lys-C in E. coli.
• To identify factors influencing the recovery of active Lys-C enzyme.

Main Methods:

• Codon-optimized Lys-C gene cloned into pET26b (+) for periplasmic expression in E. coli Rosetta (DE3).
• Investigated IPTG concentration, cell density, reducing agents (glutathione, cysteine, 2-mercaptoethanol), and harvesting time.
• Purified Lys-C using DEAE and Ni-NTA chromatography.

Main Results:

• Highest expression at 0.05 mM IPTG (5.49%).
• Optimal conditions (8 mM cysteine, induction at OD600: 0.45, 6h incubation) increased activity by 23.5%, 13.3%, and 76.4%.
• Reducing agents in media and buffers significantly enhanced Lys-C activity (up to 81.6%) and improved folding.

Conclusions:

• Optimized periplasmic expression and purification significantly increased Lys-C activity.
• Reducing agents are critical for proper Lys-C folding and enhanced enzyme function.
• This study provides a robust method for producing active recombinant Lys-C for proteomics.