High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies.
Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective expression system in Escherichia coli for the production of ET-1 enriched with (15)N and (13)C isotopes. Employing thioredoxin as carrier protein, specific and nearly quantitative cleavage of ET-1 from the fusion was mediated by Factor Xa, and purification to homogeneity (final purity of >95%) was achieved by RP-HPLC. Purified recombinant ET-1 was found to be indistinguishable from the synthetic counterpart as determined by mass spectrometry and NMR spectroscopy. Our expression strategy offers the potential for production of isotopically labeled ET-1 in large (mg) quantities for the purpose of heteronuclear NMR experiments. Moreover, the method devised should be applicable for recombinant expression of small peptides in general.
Abt. NMR-unterstützte Strukturbiologie, Forschungsinstitut für molekulare Pharmakologie, Berlin, Germany.
Thien-Thi
TT
Mac
Michael
M
Beyermann
José Ricardo
JR
Pires
Peter
P
Schmieder
Hartmut
H
Oschkinat
D004591:Electrophoresis, Polyacrylamide Gel; D019332:Endothelin-1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019332:Endothelin-1; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins