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

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.

Characterization and Immunogenicity of HIV Envelope gp140 Zera® Tagged Antigens

“…These results are in accordance with a previous study which showed that, unlike zeolin-Nef that formed PBs, zein-Nef did not efficiently form PBs and could not be distinguished from the ER (de Virgilio et al, 2008). Zein-rhBMP2ad also did not form protein bodies but was retained in the ER (Ceresoli et al, 2016). Our findings present another case where Zera R -tagged gp140 did not efficiently form PBs but was associated with the ER, thereby separating and settling in fractions of densities previously noted where ER membrane fragment settles.…”

“…The authors did not think the failure to form PBs was due to misfolding that could be caused by the possible aberrant disulfide bridges (hBMP2ad contains 7 Cys residues). Instead, they reasoned that the failure of zein-rhBMP2ad to form PBs may have been due to N-glycosylation that increased the solubility of rhBMP2ad and prevented PB formation (Ceresoli et al, 2016). We note that gp140 contains ≈28 N-glycosylation sites which could favor solubility of the Zera R -tagged gp140, thus preventing optimal assembly into PBs.…”

“…Thus, we reasoned that if aberrant disulfide bond formation is a common occurrence for recombinant gp140 proteins, fusion to the Cys-rich Zera R coding sequence would probably exacerbate this phenomenon. Ceresoli et al (2016) reported another case where zeintagged recombinant human bone morphogenetic protein 2 active dimers (zein-rhBMP2ad) accumulated at higher levels than the native soluble protein (hBMP2nat) but failed to induce PBs. The authors did not think the failure to form PBs was due to misfolding that could be caused by the possible aberrant disulfide bridges (hBMP2ad contains 7 Cys residues).…”

“…It should be noted, however, that not all antigens of interest fused to zein accumulate in PBs (de Virgilio et al, 2008;Ceresoli et al, 2016), an indication that the properties of the protein of interest need careful consideration to increase the likelihood of benefiting from properties inherent to zein PBs.…”

Where do Protein Bodies of Cereal Seeds Come From?

“…27 kD γ-zein is composed of an N-terminal domain characterized by eight repeats of the amphipathic sequence ValHisLeuProProPro and seven Cys residues, followed by a C-terminal 8CM/ABC domain. The N-terminal domain promotes PB formation when fused to other proteins that are otherwise available for intracellular traffic ( Mainieri et al, 2004 ; Llop-Tous et al, 2010 ), although this dominant effect is not universal ( de Virgilio et al, 2008 ; Ceresoli et al, 2016 ). Deletion of the N-terminal domain from γ-zein causes secretion of the remaining ABC domain, and the reciprocal deletion causes ER retention of the N-terminal domain, although in this case the typical round-shaped PBs are not formed ( Geli et al, 1994 ).…”