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

Chenhui Chen; Ziye Zhao; Jin XU; Xuesong Cao; Shangjing Guo; Jun LI; Hao Wang; Sheng Hou

Return

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

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

1. School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
2. School of Life Sciences, Liaocheng University, Liaocheng 252000, Shandong, China.
3. Shanghai Zhangjiang Biotechnology Co., Shanghai 201203, China.
4. School of Pharmacy, Liaocheng University, Liaocheng 252000, Shandong, China.
5. Cancer Institute, the Second Military Medical University, Shanghai 200433, China.
Publication Type:Journal Article
Keywords: atrial natriuretic peptide; peptides purification; tandem expression
MeSH: Atrial Natriuretic Factor; biosynthesis; Electrophoresis, Polyacrylamide Gel; Escherichia coli; metabolism; Gene Expression; Humans; Metalloendopeptidases; Peptides; Plasmids; genetics; Recombinant Fusion Proteins; biosynthesis
From: Chinese Journal of Biotechnology 2016;32(9):1273-1285
CountryChina
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.