title: Purification by immobilized metal affinity chromatography of human atrial natriuretic peptide expressed in a novel thioredoxin fusion protein.
abstract: A fusion protein that contains human atrial natriuretic peptide (ANP) at its carboxy terminus has been genetically engineered with the objective of being able to produce the peptide in a process with a relatively simple purification procedure. The fusion protein also includes a (His)6 metal affinity binding site at the amino terminus, followed by Escherichia coli thioredoxin, a factor Xa protease recognition site, and ANP. With induction of the tac promoter at 30 degrees C, the expression level of the fusion protein was high (10% of total cell protein as measured by densitometry) and it was almost completely (92%) expressed as a soluble protein in the cytoplasm. A step gradient elution with imidazole of a column of Ni2+ chelated to iminodiacetic acid-agarose saturated with proteins in crude cell extract gave a very nearly pure fusion protein. After digestion of the purified fusion protein with factor Xa protease, ANP of exactly the correct size (to within 2 Da) was observed by coupled HPLC/mass spectrometry.
authors.affiliation: School of Chemical Engineering and Materials Science, University of Oklahoma, Norman 73019-0628, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Wilkinson
authors.forename: N T
authors.initials: NT
authors.lastname: Ma
authors.forename: C
authors.initials: C
authors.lastname: Haught
authors.forename: R G
authors.initials: RG
authors.lastname: Harrison
mesh_terms: D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D005818:Genetic Engineering; D006801:Humans; D008670:Metals; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D013879:Thioredoxins / Q000096:biosynthesis / Q000302:isolation & purification*
chemical_list: D008670:Metals; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D009320:Atrial Natriuretic Factor