The pro-sequence facilitates folding of human nerve growth factor from Escherichia coli inclusion bodies

Authors


E. Schwarz, Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle/Saale, Germany. Fax: + 49 345 55 27013, Tel.: + 49 345 55 24856, E-mail: elisabeth.schwarz@biochemtech.uni-halle.de

Abstract

Nerve growth factor (β-NGF), a neurotrophin required for the development and survival of specific neuronal populations, is translated as a prepro-protein in vivo. While the presequence mediates translocation into the endoplasmic reticulum, the function of the pro-peptide is so far unknown. As the pro-sequences of several proteins are known to promote folding of the mature part, the renaturation behaviour of recombinant human β-NGF pro-protein was compared to that of the mature form. Expression of rh-pro-NGF in Escherichia coli led to the formation of inclusion bodies (IBs). The presence of the covalently attached pro-sequence significantly increased the yield and rate of refolding with concomitant disulfide bond formation when compared to the in vitro refolding of mature NGF (rh-NGF). Physicochemical characterization revealed that rh-pro-NGF is a dimer. The pro-peptide could be removed by limited proteolysis with trypsin yielding biologically active, mature rh-NGF. Furthermore, rh-pro-NGF exhibited biological activity in the same concentration range as rh-NGF.

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