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 objec tive of being able to produce the peptide in a process with a relative ly 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 X(a) protease recognition site , and ANP. With induction of the tac promoter at 30 degrees C, the exp ression level of the fusion protein was high (10% of total cell protei n as measured by densitometry) and it was almost completely (92%) expr essed as a soluble protein in the cytoplasm. A step gradient elution w ith imidazole of a column of Ni2+ chelated to iminodiacetic acid-agaro se saturated with proteins in crude cell extract gave a very nearly pu re fusion protein. After digestion of the purified fusion protein with factor X(a) protease, ANP of exactly the correct size (to within 2 Da ) was observed by coupled HPLC/mass spectrometry.