High yield expression and purification of human endothelin-1.
A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 micrograms/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 micrograms of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%.
Fassina
G
G
Protein Engineering Unit, TECNOGEN S.c.p.A., Piana di Monte Verna (CE), Italy.
Merli
S
S
Germani
S
S
Ciliberto
G
G
Cassani
G
G
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003001:Cloning, Molecular; D010450:Endopeptidases; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis* / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000096:biosynthesis* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D019332:Endothelin-1; D016232:Endothelins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D010450:Endopeptidases; D002918:Chymotrypsin