Eukaryotic protein production in designed storage organelles

Abstract

Background: Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein gamma zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.

Results: Various Zera fusions with fluorescent and therapeutic proteins accumulate in induced PB-like organelles in all eukaryotic systems tested: tobacco leaves, Trichoderma reesei, several mammalian cultured cells and Sf9 insect cells. This accumulation in membranous organelles insulates both recombinant protein and host from undesirable activities of either. Recombinant protein encapsulation in these PBs facilitates stable accumulation of proteins in a protected sub-cellular compartment which results in an enhancement of protein production without affecting the viability and development of stably transformed hosts. The induced PBs also retain the high-density properties of native seed PBs which facilitate the recovery and purification of the recombinant proteins they contain.

Conclusion: The Zera sequence provides an efficient and universal means to produce recombinant proteins by accumulation in ER-derived organelles. The remarkable cross-kingdom conservation of PB formation and their biophysical properties should have broad application in the manufacture of non-secreted recombinant proteins and suggests the existence of universal ER pathways for protein insulation.

Figure 1

Proline-rich domain of γ zein (Zera) induces PB-like organelles in plants, CHO cells, insect cells and fungi. (A) Confocal image of PB-like organelles formed in epidermal leaf cells of tobacco transformed with Zera-ECFP (see a higher magnification image in the inset). (B) ER network image of ECFP retained in the ER of tobacco cells expressing SPg-ECFP-KDEL. (C) ECFP secretion pattern in tobacco cells transformed with SPg-ECFP construct. (D) PB-like organelles (arrow) in CHO cells transfected with Zera-ECFP construct. (E) ER pattern in SPgECFP-KDEL expressing CHO cells. (F) Fluorescent ER and Golgi complex (arrowheads) in SPgECFP expressing CHO cells denoting ECFP secretion. (G) Immunoblot using an anti-GFP antibody of cell extracts and media of CHO cultured cells expressing Zera-ECFP (lanes 1), ECFP preceded by the γ zein N-terminal signal peptide (SPg-ECFP) (lanes 2) and ECFP-KDEL (from SPg-ECFP-KDEL construct) (lanes 3). CHO cells expressing both, Zera-GFP (H) and calnexin-DsRed (ER membrane marker (I) show co-localization of both proteins (merge in J). Co-expression of Zera-ECFP (K) and a glycosyltranferase labelled with YFP (Golgi reporter in yellow, L), does not result in these proteins co-localization (merge in M). (N) Hyphal filaments of Trichoderma reesei transformed with Zera-EGFP showing green fluorescent PB-like organelles within hyphae (arrow). (O, P) Baculovirus-mediated expression of DsRed (O) and Zera-DsRed (P) in Sf9 cells showing the induction of red fluorescent PBs (arrow in P).

Figure 2

Several biopharmaceutical proteins fused to Zera remain encapsulated in PB-like organelles in transfected mammalian cells. (A) Western blots of Zera-Ct, Zera-hGH and Zera-EGF demonstrating accumulation inside transfected mammalian cells (lanes 1) without significant secretion into the cell culture media (lanes 2). (B-D) Immunodetection of Zera-Ct in 293T (B), Cos1 (C) and CHO (D) transfected cells using an anti-calcitonin antibody and FITC-labeled secondary antibody. Green fluorescence indicates recombinant protein accumulation in PB-like organelles in all three cases. (E, F) Electron microscopy images of a CHO cell line stably transfected with Zera-hGH; (E) CHO cell showing a PB-like organelle (PB) containing electron-dense structures surrounded by a classical rough-ER membrane (see ribosomes in inset). (F) Immunodetection of Zera-hGH recombinant protein in PBs induced in transfected CHO cells. Cryosections were incubated with an anti-hGH antibody and labeled with Protein A-gold particles (10 nm). PB, protein body-like organelle; M, mitochondria; ER, endoplasmic reticulum. (G) Viability of CHO cells stably transfected with Zera-ECFP and Zera-EGF. Relative percentages of living, necrotic and apoptotic transfected cells were compared to those of non-transfected controls (CHO WT). (H) Proliferation profiles of CHO cells expressing Zera-hGH and Zera polypeptides as compared to non-transfected CHO WT cells over 1 to 4 days of incubation.

Figure 3

Zera stabilizes biopharmaceutical proteins in transformed plants. (A) Immunodetection with anti-Zera antibody (anti-R8) of Zera-EGF (lane 1) and Zera-Ct (lane 2) in leaves of stably-transformed tobacco plants. Arrows indicate oligomers, and C denotes lanes containing proteins from non-transformed controls. (B) Stability of Zera-EGF and Zera-Ct fusion proteins in desiccated leaves of transgenic tobacco plants. Protein extracts from equivalent amounts of tissue were loaded in each lane and relative accumulation was visualized by anti-Zera immunoblotting of protein extracts from fresh (F) and desiccated (D) leaves. (C-E) Immunogold labeling of Zera-Ct inside newly formed PBs in transformed tobacco leaves using anti-R8 (C), anti-calcitonin (D) and anti-BiP (E) antibodies. PB, induced PBs.

Figure 4

Sf9 insect cells accumulate high levels of Zera fusions in induced PBs. (A-C) Electron micrographs of control and Zera-EGF Sf9 cells. (A) Cells immediately post-infection. (B) Electron-dense PB-like structures (PB) visible at 2 days post-infection. (C) Immunogold labeling of Zera-EGF expressing Sf9 cells using anti-EGF antisera. N: nuclei; M: mitochondria; ER. endoplasmic reticulum; PB: PB-like organelles. (D) Silver stained SDS-PAGE (left) and immunoblot (right) of protein extracts from Sf9 cells expressing Zera-EGF (lanes 2), expressing EGF alone (lanes 1) and non-infected control cells (lanes C). Note the high accumulation of Zera-EGF in lanes 2 (arrows) compared with equivalent non-Zera EGF-expressing cell extracts (lane 1, arrowhead).

Figure 5

PBs isolation by density and Zera-fusion proteins recovery. (A-C) Protein analysis of fractions collected from a sucrose gradient of Zera-EGF tobacco leaf homogenates. (A) Immunoblot (αR8 antibody) shows Zera-EGF primarily in the dense F3 fraction (1.18 to 1.26 g/cm³ interface, arrow). (B) Immunoblot using anti-BiP antibody indicates that the soluble ER chaperone co-sediments with Zera-EGF in the F3 fraction. (C) Silver stained SDS-PAGE of sucrose fractions showing the enrichment of Zera-EGF in the F3 fraction. (D) Subcellular fractionation of homogenates of T. reesei expressing Zera-GFP (right) and GFP-KDEL (left). The analysis of fractions by Coomassie staining shows highly concentrated Zera-GFP in the dense fraction F3 (1.18 to 1.26 g/cm³ interface, arrow). (E) Protein analysis by gel electrophoresis and silver stain of fractions collected from sucrose gradients of Zera-EGF (left) and EGF (right) Sf9 expressing cells. Recombinant Zera-EGF fusion is concentrated in the dense fraction F3 (arrow) where PBs sediment.