Prokaryotic expression, purification and identification of recombinant human atrial natriuretic peptide / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1273-1285, 2016.
Artigo
em Chinês
| WPRIM
| ID: wpr-310540
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Peptídeos
/
Plasmídeos
/
Proteínas Recombinantes de Fusão
/
Metaloendopeptidases
/
Expressão Gênica
/
Fator Natriurético Atrial
/
Eletroforese em Gel de Poliacrilamida
/
Escherichia coli
/
Genética
/
Metabolismo
Tipo de estudo:
Estudo prognóstico
Limite:
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2016
Tipo de documento:
Artigo
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