[Use of Ssp dnaB mini-intein as a fusion partner for preparation of recombinant human brain natriuretic peptide].
Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.
Di
Lie
L
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China.
Zhang
Hong-Wei
HW
Xu
Lang-Lai
LL
D000818:Animals; D053488:DnaB Helicases / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D047668:Inteins / Q000235:genetics*; D020097:Natriuretic Peptide, Brain / Q000096:biosynthesis* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D019154:Protein Splicing; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D020097:Natriuretic Peptide, Brain; D053488:DnaB Helicases