OBJECTIVE: To enhance the production and bioactivity of recombinant human disintegrin domain of ADAM15 (A Disintegrin and Metalloproteinase 15) (rhADAM15) in Escherichia coli. METHODS: The expression host was chose and the recombinant expression plasmid pGEX-ADAM15 was constructed, based on analysis of the cDNA sequence of rhADAM15. RESULTS: (1) 298 mg/L GST (Glutathione-S-transferase)-ADAM15 and 42 mg/L rhADAM15 were achieved when choosing E. coli Rosetta (DE3) as the expression host that could supply additional tRNA for rare codons. (2) The GST-ADAM15 expression level increased to 326 mg/L after changing the rare codon GGA (Gly425) to GGC by PCR (Polymerase Chain Reaction)-based site-directed mutagenesis. (3) The rhADAM15 concentration increased to 57 mg/L by deleting the "Pro-Glu-Phe" at the GST-ADAM15 junction to improve the thrombin cleavage efficiency. (4) Finally, by combinational introduction of the favorable codons and suitably eliminating of certain amino acid sequence, rhADAM15 concentration reached the highest level (68 mg/L). CONCLUSION: The high expression of heterologous protein could be achieved by releasing rare codon usage and amino acids residues restriction.
OBJECTIVE: To enhance the production and bioactivity of recombinant human disintegrin domain of ADAM15 (A Disintegrin and Metalloproteinase 15) (rhADAM15) in Escherichia coli. METHODS: The expression host was chose and the recombinant expression plasmid pGEX-ADAM15 was constructed, based on analysis of the cDNA sequence of rhADAM15. RESULTS: (1) 298 mg/L GST (Glutathione-S-transferase)-ADAM15 and 42 mg/L rhADAM15 were achieved when choosing E. coli Rosetta (DE3) as the expression host that could supply additional tRNA for rare codons. (2) The GST-ADAM15 expression level increased to 326 mg/L after changing the rare codon GGA (Gly425) to GGC by PCR (Polymerase Chain Reaction)-based site-directed mutagenesis. (3) The rhADAM15 concentration increased to 57 mg/L by deleting the "Pro-Glu-Phe" at the GST-ADAM15 junction to improve the thrombin cleavage efficiency. (4) Finally, by combinational introduction of the favorable codons and suitably eliminating of certain amino acid sequence, rhADAM15 concentration reached the highest level (68 mg/L). CONCLUSION: The high expression of heterologous protein could be achieved by releasing rare codon usage and amino acids residues restriction.