EXPRESSION OF THE HUMAN COMPLEX-FORMING GLYCOPROTEIN HC (ALPHA-1-MICROGLOBULIN) IN ESCHERICHIA-COLI

The mature form of human protein HC, or alpha 1-microglobulin, has bee n expressed in Escherichia coli. Protein HC is a member of the lipocal in superfamily of hydrophobic ligand-binding proteins, and carries a h eterogeneous chromophore linked covalently by a reduction-resistant bo nd. Protein HC was first overexpressed as a C-LytA/HC fusion protein c ontaining the C-terminal moiety of the pneumococcal lytic amidase (Lyt A). Recombinant C-LytA/HC was found to be an insoluble aggregate that was solubilized with 6 M guanidinium chloride and renatured by the add ition of thiol reagents in the presence of L-arginine. Recombinant pro tein HC (rHC) was released from C-LytA/HC by trypsin digestion and pur ified by size-exclusion chromatography. rHC protein possesses an N-ter minal amino-acid sequence identical to that of human protein HC, and a slightly lower molecular mass as determined by SDS-PAGE. Both C-LytA/ HC and rHC reacted with polyclonal antibodies raised against native pr otein HC. A photodiode array detection system on-line with a HPLC syst em has allowed the identification of a chromophore associated to rHC p rotein displaying significant absorption in the visible region of the spectrum in resemblance to that found in the natural form of human pro tein HC.