Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli.
The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.
Becker
A
A
Research Laboratories of Schering AG, Berlin, Germany.
Theuring
F
F
Gottwald
M
M
Kauser
K
K
Schleuning
W D
WD
Donner
P
P
D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017364:Collagenases; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D016232:Endothelins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
D016232:Endothelins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D017364:Collagenases