Effect of zinc binding and precipitation on structures of recombinant human growth hormone and nerve growth factor

Metal-induced precipitation of protein therapeutics is being used and furth er developed as a processing step in protein formulation and may have utili ty in protein purification and bulk storage. In such processes, it is imper ative that native protein structure is maintained and the metal complexatio n is reversible. In the current study, we investigated the effects of zinc- induced precipitation on recombinant human growth hormone (rhGH) and recomb inant human nerve growth factor (rhNGF). On the addition of ethylenediamine tetraacetic acid (EDTA), the precipitates were dissolved, yielding complete recovery of native protein in both cases. Both proteins have specific meta l binding sites and require specific molar ratios of zinc to protein to ini tiate precipitation (zinc:rhGH > 2:1; zinc:rhNGF > 18:1). Furthermore, the secondary structures of both proteins were unperturbed in soluble zinc comp lexes and zinc-induced precipitates, as measured by infrared and circular d ichroism spectroscopies. The soluble zinc complex of rhGH had minor tertiar y structural alterations, whereas zinc binding did not alter the tertiary s tructure of rhNGF. These studies indicated that metal-induced precipitation provides a method to maintain proteins in their native state in precipitat es, which may be useful for purification, storage, and formulation. (C) 200 0 Wiley-Liss, Inc. and the American Pharmaceutical Association.