RECOMBINANT HUMAN CHROMOGRANIN-A - EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE N-TERMINAL DERIVED PEPTIDES

Chromogranin A (CGA) is an ubiquitous 48 kDa secretory protein stored and released from most endocrine cells and is present in nanomolar concentration in the human vascular system. Recent data suggest that CGA may be the precursor of several peptides with a defined biological activity. The present report describes the expression of human CGA in Escherichia coli using the pET3a vector system, the purification and characterization of the recombinant protein and the production of antibody against the expressed protein. The expressed CGA was purified by a multi-step protocol including heat treatment, gel filtration and high performance-anion exchange chromatography and two-dimensional gel electrophoresis. Two major forms of recombinant human CGA (rhCGA) were purified from the bacterial cytosol: a 70 kDa form which corresponded to the native full-length CGA and a major proteolytic 63 kDa product recognized by antibodies raised against the 70 kDa rhCGA or to synthetic peptides localized in the N-terminal part of the bovine CGA sequence. This E. coli expression system provides a method for producing a suitable protein which will permit the identification of CGA-derived peptides with defined biological function in human. Fragments containing the N-terminal domain were generated by acidic cleavage of the two forms of rhCGA. A two-step purification using high-performance reverse-phase chromatography yielded 6 peptide bands ranging in apparent molecular mass from 7 to 18 kDa. Four components (molecular mass range 12-18 kDa) were immunostained with antibodies directed against synthetic sequences of bovine vasostatin II (bCGA(1-113)) while the two others (molecular mass range 7-8 kDa) were immunostained only with antibodies directed against vasostatin I (bCGA(1-76)). From protein staining the ratio vasostatins II/I was 10:1. The vasoinhibitory activity of this preparation was examined on isolated human saphenous vein segments. An inhibitory effect was obtained in paired vessel segments from 7 patients undergoing surgery for coronary artery bypass, however with low potency for supression of the endothelin-1 evoked sustained tension in these vessels.