Chinese Journal of Biotechnology 2016;32(9):1273-1285

doi:10.13345/j.cjb.160018

Prokaryotic expression, purification and identification of recombinant human atrial natriuretic peptide.

Chenhui CHEN 1 ; Ziye ZHAO 2 ; Jin XU 3 ; Xuesong CAO 2 ; Shangjing GUO 4 ; Jun LI 4 ; Hao WANG 5 ; Sheng HOU 5

Affiliations

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Keywords

atrial natriuretic peptide; peptides purification; tandem expression

Country

China

Language

Chinese

Abstract

In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.