ABSTRACT
Nerve growth factor (beta-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 beta-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.
Subject(s)
Escherichia coli/metabolism , Nerve Growth Factor/chemistry , Protein Precursors/chemistry , Electrophoresis, Polyacrylamide Gel , Electroporation , Escherichia coli/genetics , Humans , Inclusion Bodies/metabolism , Nerve Growth Factor/biosynthesis , Nerve Growth Factor/genetics , Plasmids , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/chemistry , UltracentrifugationABSTRACT
Nerve growth factor (NGF) is a member of the neurotrophin family. These growth factors support neuronal survival and differentiation. Neurotrophins are synthesized as pre-pro-proteins. Whereas the pre-sequences mediate secretion, the function of the pro-peptides is largely unknown. To test the role of the pro-sequence as a folding enhancer, recombinant human pro-NGF (rh-pro-NGF) was produced in Escherichia coli. The oxidative refolding of rh-pro-NGF and rh-NGF was studied using electrospray mass spectrometry (ESIMS) time-course analysis. This analysis permitted both the identification and quantification of intermediates present during the process. The disulfide bonds formed at different times of the refolding processes were characterized by proteolytic digestion followed by matrix assisted laser desorption ionization mass spectrometry (MALDIMS) analysis. Folding yields and kinetics of rh-pro-NGF were significantly enhanced when compared to the in vitro refolding of mature rh-NGF. These results suggest that the pro-sequence of NGF promotes folding of the mature part.
