Expression and purification of active recombinant equine lysozyme in Escherichia coli.

The research involves the successful creation and purification of functional equine lysozyme, a calcium-binding protein, in the bacterium Escherichia coli. This process allows for the investigation of the protein’s structure, function, and ability to form specific fibrils, which are of interest in molecular biology.

Overview of the Research

• The research revolves around equine lysozyme (EL), a calcium-binding protein that represents a connecting link between non-calcium-binding C-type lysozyme and alpha-lactalbumin. The study is driven by an interest in these proteins’ capability to assemble into fibril structures, formations that are currently gaining significant attention in molecular biology.
• To delve deeper into EL’s functional and structural properties, the researchers synthesized a gene that was later cloned. The overexpression of this gene in Escherichia coli (E. coli) led to the creation of fused EL protein.

Recombinant EL Creation and Purification

• The recombinant equine lysozyme (rEL), featuring His-tagged markers, was stored as inclusion bodies within the E. coli.
• The recombinant EL was extracted and purified through a multi-step process. This involved using Ni(2+) affinity chromatography, which separates biomolecules based on their various binding affinities, and refolding the extracted protein in the presence of arginine, an amino acid that helps in protein refolding.
• Following this, CM-Sepharose column purification was applied after the cleavage by TEV protease. The resulting yield offered up to 50mg per litre of purified, recombinant EL.

Study of Purified Protein

• The purified protein remained functional, evidenced by lysozyme activity that validated the protein’s correct folding.
• The team used this purified protein for oligomerisation studies, which analyze protein complex formations like fibrils.
• The results showed that, when incubated under high temperature and acidic pH conditions, the protein formed amyloid fibrils – particular proteins that can form in several ways and are of interest due to their connection to several diseases.
• The recombinant protein was found to form two types of fibrils: ring-shaped and linear, similar to the native EL.

Implications of the Research

• These findings not only demonstrate a process to create and purify active rEL in a bacteria like E. coli, but also shed light on the properties and behaviors of this protein under specific conditions.
• The research also opens the door for further studies on the role and workings of such proteins and fibrils in molecular biology and related fields, contributing to a greater understanding of these protein structures and their correlation to various diseases.