Codon optimization for high level expression of human bone morphogenetic protein-2 in Escherichia coli

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Engineering

Background:

• Human bone morphogenetic protein-2 (hBMP-2) is crucial for bone regeneration.
• Efficient recombinant production of hBMP-2 is essential for therapeutic applications.
• Optimizing codon usage in E. coli enhances protein expression.

Purpose of the Study:

• To optimize codons for high-level expression of human bone morphogenetic protein-2 (hBMP-2) in Escherichia coli.
• To develop a robust protocol for the production and purification of recombinant hBMP-2.
• To validate the Thermodynamically Balanced Inside Out (TBIO) approach for protein expression optimization.

Main Methods:

• Codon optimization of the hBMP-2 open reading frame (ORF) using DNAWorks and DNAStar software.
• Gene assembly via two-step Polymerase Chain Reaction and cloning into pGEM-T vector.
• Transformation into E. coli strains JM109 and BL21(DE3) for expression in pET32b vector.
• Purification of recombinant hBMP-2 (rhBMP-2) as a thioredoxin-his-tag fusion protein from inclusion bodies.
• Solubilization, refolding, and characterization of rhBMP-2 using nano-LC-MS/MS2 analysis.

Main Results:

• Codon optimization resulted in high-level expression of rhBMP-2, constituting approximately 60% of total intracellular proteins as inclusion bodies.
• A yield of 1.39 g/L of culture was achieved.
• Solubilization and refolding processes yielded monomeric and dimeric forms of rhBMP-2 with recovery rates of 13.6% and 8.7%, respectively.
• The identity and size of the purified rhBMP-2 were confirmed by nano-LC-MS/MS2.

Conclusions:

• The Thermodynamically Balanced Inside Out (TBIO) approach enables high-level expression of codon-optimized hBMP-2 in E. coli.
• This study presents a successful strategy for producing functional recombinant hBMP-2.
• The optimized expression system provides a foundation for large-scale production of rhBMP-2 for therapeutic use.