Use of Ssp dnaB derived mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli.
To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, a modified Ssp dnaB mini-intein linked with a chitin binding domain tag was used as a fusion partner for production of human brain natriuretic peptide (hBNP), a hormone for the treatment of congestive heart failure. The fusion protein was expressed as an inclusion body in Escherichia coli. After refolding, the fusion protein was purified with a chitin affinity column, and dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH in the chitin column to 7.0 at 25 degrees C for 16 h, which led to the release and separation of hBNP from its fusion partner. The hBNP sample was further purified with reverse phase HPLC and its biological activity was assayed in vitro. It was found that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC(50) of (1.24+/-0.32)x10(-6)mg/ml, which was similar to that of the synthetic BNP standard. The expression strategy described here promises to produce small peptides without use of proteolytic or chemical reagents.
Sun
Ziyong
Z
Institute of Molecular Medicine, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, 210093 Nanjing, China.
Chen
Junyong
J
Yao
Hongwei
H
Liu
Lili
L
Wang
Jing
J
Zhang
Jing
J
Liu
Jian-Ning
JN
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D004265:DNA Helicases / Q000737:chemistry; D053488:DnaB Helicases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D050939:Gene Fusion / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D047668:Inteins; D008969:Molecular Sequence Data; D020097:Natriuretic Peptide, Brain / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D020097:Natriuretic Peptide, Brain; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; C506730:dnaB protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases