STRUCTURAL CHARACTERIZATION OF NATIVE AND RECOMBINANT FORMS OF THE NEUROTROPHIC CYTOKINE MK

The retinoic acid (RA)-inducible midkine (MK) gene encodes a heparin-b inding protein which can induce neurite outgrowth in cultured mammalia n embryonic brain cells. This cytokine shares 65% amino acid sequence identity with another RA-inducible cytokine, pleiotropin (PTN). Both p roteins contain 10 conserved cysteine residues, all of which appear to be disulphide linked. MK and PTN are also rich in lysine and arginine residues rendering them susceptible to proteolysis during purificatio n, and making large-scale preparation of these molecules inherently di fficult. Recombinant MK has been expressed as a fusion protein using a pGEX vector transfected into E. coli. To enable refolding of MK, the fusion protein was stored in solution at 4-degrees-C for 14 days in th e presence of dithiothreitol (DTT). Thrombin cleavage of the fusion pr otein, post storage, typically generated 5 mg of MK per litre of bacte rial pellet. To establish the structural integrity of the recombinant product, we have analysed the refolding kinetics and compared the disu lphide bond assignment of recombinant MK with that of native MK and na tive PTN. The synergistic use of micropreparative HPLC, to separate an d recover in small eluant volumes enzymatically derived peptide fragme nts, with matrix assisted laser desorption mass spectrometry (MALD-MS) and N-terminal sequence analysis has allowed the unambiguous identifi cation of the disulphide bonded fragments of native and recombinant MK . The disulphide bond assignment of MK is C-12-C36, C2O-C45, C27-C49, C59-C91 and C69-C101, and is equivalent to that of PTN.