Purification by immobilized metal affinity chromatography of human atrial natriuretic peptide expressed in a novel thioredoxin fusion protein.

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.