Plant Seed Oil-bodies as Carriers for Foreign Proteins

Abstract

Plant seeds frequently store oils (triglycerides) in discrete organelles called oil-bodies. These are normally surrounded by a phospholipid half-unit membrane equipped with specialized proteins called oleosins. Oleosins are highly lipophilic proteins, are expressed at high levels in many seeds and are specifically targeted to oil-bodies. We have investigated the potential of oleosins to act as carriers for recombinant proteins by the production of translational fusions between oleosins and genes encoding proteins foreign to plant cells. We have shown that a fusion comprising a complete oleosin coding domain and a beta-glucuronidase coding sequence may be expressed specifically in the seeds of the oilseed crop plant, Brassica napus, and its product is correctly targeted with approximately 80% of the activity partitioning with oil-bodies. Recombinant oil-bodies may be used to facilitate separation of a recombinant protein from other cellular proteins. Using this approach, the desired protein may be cleaved from the oil-bodies using an endoprotease and further purified. Alternatively, a fusion protein which is enzymatically active and resides on the oil-bodies may be used directly in heterogeneous catalysis. In this application, after a round of catalysis the oil-bodies may be recovered and re-used several times without loss of activity. Thus the oil-bodies act as an immobilization matrix. The fusion protein is stable in dry seeds for long periods and when extracted has a half-life of 3-4 weeks on oil-bodies. Finally, the production of these recombinant oil-bodies is extremely inexpensive, offering a novel route to the manufacture of recombinant proteins.

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

“…We used total chloroplasts as the starting material in the purification procedure, because of the association of PGL35-HIVp24 with both the plastoglobule and thylakoid fractions. For this reason also, purification protocols resembling the one in the oleosin system (Vanrooijen and Moloney 1995;Boothe et al 2010) using a flotation centrifugation step may be inefficient for most plastoglobulin fusion proteins. It is possible however that PGL35 fusion proteins that are more hydrophobic and have a lower isoelectric point may accumulate to a high degree in plastoglobules and be more readily purified by flotation centrifugation.…”

Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A

“…Several potential biotechnological solutions have been described in the literature that propose fusing protein A (or other affinity ligands) to elements capable of polymerizing (bacteriophage capsid proteins [26][27][28]) or binding to structures of high molecular weight (bacterial S-layer proteins [29,30], oleosins recognizing oil bodies [31,32], cellulose-binding domains [33,34], starch-binding domains [35]). So far, only the process based on oleosin fusions (to protein A) has been advanced to the stage of a commercially viable platform (www.sembiosys.com).…”

Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain

“…S1OLEO and S2OLEO were created using primer OSS1 (5Ј-CCGGACGAGGATCTGACTACTCCGGATCCGGGCAGATT-GCTGGAGCTGCAACTGCTGTCACAGCTGG-3Ј; BamHI site underlined) and OSS2 (5Ј-CCAAGTCTAGGCAGATTGCTAAATCTTCAA-CTAGTTCCACAGCTGGTGGTTCCCTCC-3Ј; SpeI site underlined), respectively, by the Kunkel method of site-directed mutagenesis (31). ␤-glucuronidase was used in pGNOS (22). OSS3 was generated from WTOLEO by PCR using primers OSS3a and OSS3b (5Ј-CTGCTCCAT-GGATGTTGCAACAGTCAAAGCTA-3Ј; NcoI site underlined) and then cloned at the 5Ј end of GUS by fusion of the common NcoI sites to yield OSS3G.…”

“…It is likely that membrane topology and the hydrophobicity of the H domain favors transition into a region of oil in the ER membrane from which oil body formation can occur (18 -20). Oleosins are also used as carriers for recombinant protein production in seeds (21,22). Thus, the topology of this protein has a significant impact on the potential uses of oleosin-based production systems.…”