A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease

Area of Science:

• Biotechnology
• Protein Biochemistry
• Molecular Biology

Background:

• Recombinant protein purification is a critical bottleneck in biotechnology and medicine.
• Existing purification methods can be complex and inefficient.

Purpose of the Study:

• To develop a simple and efficient method for recombinant protein purification.
• To utilize a self-assembling peptide-tagged tobacco etch virus protease (TEVp) for purification.

Main Methods:

• Constructed an N-terminal ELK16 peptide fusion expression vector for ELK16-TEVp in E. coli.
• Purified ELK16-TEVp using centrifugation with Triton X-100.
• Assessed protease activity using His-tagged bovine interferon-γ (His-BoIFN-γ).
• Demonstrated removal of ELK16-TEVp by single-step centrifugation.
• Purified recombinant bovine interferon-γ (rBoIFN-γ) after His-tag removal.

Main Results:

• ELK16-TEVp was expressed as active protein aggregates with 91% purity and 92% recovery.
• ELK16-TEVp exhibited high protease activity (1.3 × 10^4 units/mg) and cleavage efficiency (>99%).
• Purified rBoIFN-γ achieved 98.3% purity with 63% recovery and retained antiviral activity (1.6 × 10^3 units/mg).

Conclusions:

• The ELK16-TEVp system provides an efficient and simple approach for recombinant protein purification.
• The method allows for easy removal of the protease tag, simplifying downstream processing.
• This technique holds potential as a universal tool for diverse recombinant protein purification applications.