Purification by Immobilized Metal Affinity Chromatography of Human Atrial Natriuretic Peptide Expressed in a Novel Thioredoxin Fusion Protein

摘要

AbstractA 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)6metal affinity binding site at the amino terminus, followed byEscherichia colithioredoxin, a factor Xaprotease recognition site, and ANP. With induction of thetacpromoter at 30 °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 Xaprotease, ANP of exactly the correct size (to within 2 Da) was observed by coupled HPLC/mass spectromet