A Fusion between Domains of the Human Bone Morphogenetic Protein-2 and Maize 27 kD γ-Zein Accumulates to High Levels in the Endoplasmic Reticulum without Forming Protein Bodies in Transgenic Tobacco.

Valentina Ceresoli,Roberto Weinstein,Emanuela Pedrazzini,Davide Mainieri,Massimo Del Fabbro

doi:10.3389/fpls.2016.00358

Abstract

Human Bone Morphogenetic Protein-2 (hBMP2) is an osteoinductive agent physiologically involved in bone remodeling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad) in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs.

Highlights

Throughout the adult life, as well as in the healing process following injuries, bone tissue is subjected to cycles of absorption and formation during the physiological modeling and remodeling
A single chimeric construct including the N-Terminal of 27 kDa γ-zein, a DNA linker, a Thrombin cleavage site, the complete coding sequence of human BMP2 and a FLAG epitope was purchased from GeneCust (GeneCust Europe, Laboratoire de Biotechnologie du Luxembourg S.A.) in a pUC57 vector in order to obtain the two zein-hBMP2ad and hBMP2nat constructs with specific subcloning experiments
A short flexible linker and a thrombin cleavage site were inserted between the fusion partners, to favor the independent folding of the two moieties and hBMP2ad purification (Figure 1)

Summary

INTRODUCTION

Throughout the adult life, as well as in the healing process following injuries, bone tissue is subjected to cycles of absorption and formation during the physiological modeling and remodeling. The 27 kD γ-zein (hereafter zein) is a maize storage protein belonging to the prolamin class and is able to induce PB biogenesis even when expressed in vegetative tissues of transgenic plants (Shewry et al, 1995; Vitale and Ceriotti, 2004). These polymers are insoluble unless treated with reducing agents (Mainieri et al, 2004, 2014; Pompa and Vitale, 2006) This domain has been used to allow accumulation of recombinant fusion proteins both in transgenic plants and in mammalian cells (Mainieri et al, 2004; Llop-Tous et al, 2011). In order to investigate new cost-effective approaches to express the hBMP2 in tobacco transgenic plants, we produced a chimeric protein (named zein-hBMP2ad), consisting of the C-terminal active domain of hBMP2 fused to the N-terminal domain of 27 kD γ-zein. We show that the prolamin domain promotes higher accumulation level of hBMP2ad compared to the native hBMP2 (hBMP2nat). zein-hBMP2ad assembly, post-translational modifications and ability to induced PB biogenesis were analyzed

MATERIALS AND METHODS

RESULTS

DISCUSSION