Processing of Human Nerve Growth Factor β Proprotein in the Plant Cell Apoplast

Abstract

The Human HNGF-β gene encodes a pre-pro-NGF-β precursor protein. The signal peptide is cleaved off in the endoplasmic reticulum and the resulting pro-protein is processed in the trans-Golgi network by the Furin enzyme.The processing of the proproteins in the plant cells is not clearly defined. In the present study, various changes were applied to the HNGF-β gene sequence to increase its expression in the plant cells. The construct containing the synthesized Plant NGF-β gene was agro-infiltrated into the Nicotiana benthamiana leaves. Thereafter, the expression of various forms of the recombinant NGF-β and its processing was evaluated using dot blot, western blot, and RP-HPLC analyses. Finally, the biological activity of the mature NGF-β purified from the apoplast was assessed on the differentiation of PC12 cells and the expression of tetanus toxin receptors on their surfaces.Dot blot and western blot results showed that the total soluble proteins extracted from the plant leaves and the apoplast extract contained pro-NGF-β and mature NGF-β proteins, respectively. The RP-HPLC results reconfirmed the presence of the mature NGF-β in the apoplast extract. The amount of the mature NGF-β produced in the plant leaves was estimated to be about 39µg/g of the fresh leaves. A gradual increase in the length and number of the neurites of the differentiated PC12 cells was showed upon treatment with the mature NGF-β. Immunofluorescence experiments showed that the FITC-labeled tetanus toxin strongly bound to PC12 cells treated with mature NGF-β. This modification shows a very important advantage for plants to produce valuable biosimilar pharmaceutical proteins from precursor without applying biochemical or co-expression of modifying enzymes.