Engineered β-Lactoglobulin Produced in E. coli: Purification, Biophysical and Structural Characterisation

Area of Science:

• Biochemistry
• Protein Engineering
• Structural Biology

Background:

• Bovine beta-lactoglobulin (β-Lg) is a major whey protein with potential applications.
• Production of functional recombinant β-Lg is challenging due to N-terminal processing.
• Previous methods have not yielded soluble, homogeneous, and ligand-free protein.

Purpose of the Study:

• To develop a protocol for producing functional recombinant bovine β-lactoglobulin (β-Lg).
• To characterize the biochemical and biophysical properties of the recombinant protein.
• To assess its suitability for further engineering and applications in medicine and pharmacology.

Main Methods:

• Gene engineering for E. coli expression of bovine β-Lg with L1A/I2S mutations.
• Purification using a novel protocol including Sephadex G75 gel filtration.
• Characterization via circular dichroism (CD) spectra, ligand binding assays, stability tests, and crystal structure analysis.

Main Results:

• Functional, soluble, and homogeneous recombinant bovine β-Lg was obtained.
• N-terminal mutations facilitated in vivo methionine cleavage, ensuring correct folding.
• Purified protein was nearly identical to natural β-Lg in physicochemical properties and ligand binding.
• The recombinant protein is suitable for biochemical, biophysical, and structural studies.

Conclusions:

• A robust protocol for producing functional recombinant bovine β-Lg was established.
• The engineered protein retains native-like properties and binding capabilities.
• This recombinant β-Lg is a promising candidate for advanced applications in pharmacology and medicine.
• Meta_The_study_reports_a_novel_protocol_for_producing_functional_recombinant_bovine_beta-lactoglobulin_suitable_for_medical_applications.