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

• Published in: Protein expression and purification 2012-08, Vol.84 (2), p.188-194
• Main Authors: Retnoningrum, Debbie S., Pramesti, H.T., Santika, P.Y., Valerius, O., Asjarie, S., Suciati, T.
• Format: Article
• Language: English
• Subjects: Base Sequence; Bone Morphogenetic Protein 2 - chemistry; Bone Morphogenetic Protein 2 - genetics; Bone Morphogenetic Protein 2 - isolation & purification; Cloning, Molecular - methods; Codon - genetics; Codon-optimized ORF; Escherichia coli; Escherichia coli - genetics; Gene Expression; High level expression; Human bone morphogenetic protein-2; Humans; Mass Spectrometry; Molecular Sequence Data; Open Reading Frames; Plasmids - genetics; Protein Refolding; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; TBIO
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2012.05.010

► For the first time, a codon-optimized rhBMP-2 gene was constructed using TBIO approach. ► The codon-optimized rhBMP-2 gene was expressed at high level in Escherichia coli system. ► The rhBMP-2 protein expressed from synthetic gene was confirmed by nano-LC–MS/MS2. Codons in the open reading frame (ORF)1Abbreviations used: ORF, open reading frame; TBIO, thermodynamically balanced inside out; bp, base pairs; IB, inclusion bodies; hBMP-2, human BMP-2; Tm, melting temperatures; pNPP, para nitro phenol phosphate substrate system; CAI, codon adaptation index.1 encoding for human bone morphogenetic protein-2 (hBMP-2) were optimized to reach high level expression in Escherichia coli. The optimization was done by the computer programs DNA works and DNA Star according to Thermodynamically Balanced Inside Out (TBIO) approach. The ORF consisting of 342 base pairs (bp) was assembled using two-steps Polymerase Chain Reaction, cloned into a pGEM-T vector with a mutation rate of 6.38bp per kb and transformed into E. coli JM109. After a DNA sequence confirmation, mutation-free ORF was subcloned into pET32b and transformed into E. coli BL21(DE3). The rhBMP-2 was produced as a thioredoxin-his-tag fusion protein at relatively high level, approximately 60% of total intracellular proteins as inclusion bodies (IB), with a yield of 1.39g per liter culture. Solubilization of IB gave soluble monomer rhBMP-2 with a recovery of 13.6% and refolding of soluble rhBMP-2 produced dimeric forms with a yield of 8.7%. The size and identity of the purified rhBMP-2 was confirmed by nano-LC–MS/MS2 analysis. Our work demonstrates for the first time that by using TBIO approach, a codon-optimized ORF encoding for rhBMP-2 protein can be expressed at high level in E. coli expression system.