Eukaryotic protein production in designed storage organelles

Abstract

Background: Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plantderived organelles that stably accumulate large amounts of storage proteins in seeds. The prolinerich N-terminal domain derived from the maize storage protein γ 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.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

“…The ability of a similar sequence to redirect phaseolin, a vacuolar storage protein, to the ER compartment of transformed tobacco leaves in which the fusion protein, named zeolin, is retained and accumulated in PBs, has also been demonstrated (26). Interestingly, the PBinducing capacity of Zera was also demonstrated in a variety of other (non-plant) eukaryotic cells, including mammalian, insect, and fungal cells (24). This ubiquitous behavior of Zera indicates that intrinsic molecular properties are responsible for the fusion protein assembly and that PB biogenesis is independent of specific seed or plant tissue mechanisms.…”

“…The PCR fragment replaced the original Zera sequence in pUC18ZeraECFP (24). The cassette containing the new fusion sequence Zera-ECFP was transferred to the binary vector pC2300 (see the Cambia Web site) under the control of the enhanced 35 S cauliflower mosaic virus, obtaining the plant transformation vector pCZera-ECFP (supplemental Fig.…”

“…S1). Plasmids pCSPg-ECFP and pCSPg-ECFP-KDEL were obtained as described (24). Vector pCR8-ECFP containing the repeat region sequence fused to the ECFP sequence was obtained by replacing the Zera sequence in pCZera-ECFP by a synthetic DNA sequence coding for the R8 (PPPVHL) 8 region flanked by SpeI and AscI restriction sites.…”

“…The resulting extracts were centrifuged at 10,000 ϫ g for 30 min at 4°C, and total soluble proteins were separated on 12.5% SDSpolyacrylamide gels. The proteins were detected by staining or by immunoblot using the anti-R8 antiserum (24) and anti-GFP raised in rabbits injected with a recombinant GFP protein expressed and purified from Escherichia coli cells.…”

“…These results contrast with what frequently occurs when other truncated proteins are overexpressed; they usually associate with BiP and are ultimately degraded in lytic vacuoles (19,20) or enter the ER-associated protein degradation (ERAD) pathway (21,22). The proline-rich N-terminal sequence of ␥-zein comprising the first 112 amino acids (the Zera sequence) (23) fused to a variety of reporter and therapeutic proteins, was shown to induce the stable accumulation of the fusion proteins in ER-derived PB-like structures in plant vegetative tissues (24,25). The ability of a similar sequence to redirect phaseolin, a vacuolar storage protein, to the ER compartment of transformed tobacco leaves in which the fusion protein, named zeolin, is retained and accumulated in PBs, has also been demonstrated (26).…”