Stable constitution of artificial oil body for the refolding of IGF1

Over-expression of oleosin-fused IGF1 results in the formation of insoluble aggregates in Escherichia coli occupying 35% of total proteins. In this study, a method based on artificial oil body (AOB) was applied to obtain active IGF1, insulin-like growth factor 1, from its insoluble form in one step. The stability of AOB emulsions constituted with soybean oleosin was maximized in the optimal composition of TAG (97.04%, wt/wt), PL (1.14%, wt/wt), and oleosin-UbIGF1 (1.82%, wt/wt) at pH 7.5 and at 25°C. Upon sonication, the mixture comprising plant oil and the insoluble fusion protein was readily assembled into AOBs. After peptide cleavage mediated by endopeptidase, the IGF1 free of fusion tags was liberated and then recovered. Subsequently, IGF1 self-refolded on AOB was obtained with high yield of 63.2 mg/g dry cell. This on-AOB refolding can be applied to the development of bacterial expression and purification of other active recombinant proteins.     

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

Elevation of oil body integrity and emulsion stability by polyoleosins,multiple oleosin units joined in tandem head‐to‐tail fusions

We have successfully created polyoleosins by joining multiple oleosin units in tandem head‐to‐tail fusions. Constructs encoding recombinant proteins of 1, 3 and 6 oleosin repeats were purposely expressed both in planta and in Escherichia coli. Recombinant polyoleosins accumulated in the seed oil bodies of transgenic plants and in the inclusion bodies of E. coli. Although polyoleosin was estimated to only accumulate to <2% of the total oil body protein in planta, their presence increased the freezing tolerance of imbibed seeds as well as emulsion stability and structural integrity of purified oil bodies; these increases were greater with increasing oleosin repeat number. Interestingly, the hexameric form of polyoleosin also led to an observable delay in germination which could be overcome with the addition of external sucrose. Prokaryotically produced polyoleosin was purified and used to generate artificial oil bodies and the increase in structural integrity of artificial oil bodies‐containing polyoleosin was found to mimic those produced in planta. We describe here the construction of polyoleosins, their purification from E. coli, and properties imparted on seeds as well as native and artificial oil bodies. A putative mechanism to account for these properties is also proposed.     

Stable oil bodies sheltered by a unique caleosin in cycad megagametophytes

Stable oil bodies of smaller sizes and higher thermostability were isolated from mature cycad (Cycas revoluta) megagametophytes compared with those isolated from sesame seeds. Immunological cross-recognition revealed that cycad oil bodies contained a major protein of 27 kDa, tentatively identified as caleosin, while oleosin, the well-known structural protein, was apparently absent. Mass spectrometric analysis showed that the putative cycad caleosin possessed a tryptic fragment of 15 residues matching to that of a theoretical moss caleosin. A complete cDNA fragment encoding this putative caleosin was obtained by PCR cloning using a primer designed according to the tryptic peptide and another one designed according to a highly conservative region among diverse caleosins. The identification of this clone was subsequently confirmed by immunodetection and MALDI-MS analyses of its recombinant fusion protein over-expressed in Escherichia coli and the native form from cycad oil bodies. Stable artificial oil bodies were successfully constituted with triacylglycerol, phospholipid and the recombinant fusion protein containing the cycad caleosin. These results suggest that stable oil bodies in cycad megagametophytes are mainly sheltered by a unique structural protein caleosin.     

Characterization and modelling of the hydrophobic domain of a sunflower oleosin

The oleosins are a group of hydrophobic proteins present on the surface of oil bodies in seeds, where they are thought to prevent coalescence. They contain a central hydrophobic domain of 68-74 residues that is thought to form a loop into the triacylglycerol matrix of the oil body, but the conformation adopted by this sequence is uncertain. We have therefore expressed an oleosin cDNA from sunflower (Helianthus annuus L.) in Escherichia coli as a fusion with maltose-binding protein (MBP) and isolated a peptide corresponding to the hydrophobic domain by sequential digestion with factor Xa (to remove the MBP) followed by trypsin and Staphylococcus V8 protease to remove the N- and C-terminal domains of the oleosin. Circular dichroism spectroscopy of the peptide in two solvent systems chosen to mimic the environment within the oil body (trifluoroethanol and SDS) demonstrated high proportions of alpha-helical structure, with no beta-sheet. A model was therefore developed in which the domain forms an alpha-helical hairpin structure, the two helices being separated by a turn region. We consider that this model is consistent with our current knowledge of oleosin structure and properties.     

An in vitro system to examine the effective phospholipids and structural domain for protein targeting to seed oil bodies.

An in vitro system was established to examine the targeting of proteins to maturing seed oil bodies. Oleosin, the most abundant structural protein, and caleosin, a newly identified minor constituent in seed oil bodies, were translated in a reticulocyte lysate system and simultaneously incubated with artificial oil emulsions composed of triacylglycerol and phospholipid. The results suggest that oil body proteins could spontaneously target to artificial oil emulsions in a co-translational mode. Incorporation of oleosin to artificial oil emulsions extensively protected a fragment of approximately 8 kDa from proteinase K digestion. In a competition experiment, in vitro translated caleosin and oleosin preferentially target to artificial oil emulsions instead of microsomal membranes. In oil emulsions with neutral phospholipids, relatively low protein targeting efficiency was observed. The targeting efficiency was substantially elevated when negatively charged phospholipids were supplemented to oil emulsions to mimic the native phospholipid composition of oil bodies. Mutated caleosin lacking various structural domains or subdomains was examined for its in vitro targeting efficiency. The results indicate that the subdomain comprising the proline knot motif is crucial for caleosin targeting to oil bodies. A model of direct targeting of oil-body proteins to maturing oil bodies is proposed.     

Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells

We have established a versatile method for studying the interaction of the oleosin gene product with oil bodies during oil body biogenesis in plants. Our approach has been to transiently express a green fluorescent protein (GFP)-tagged Arabidopsis oleosin gene fusion in tobacco leaf cells containing bona fide oil bodies and then to monitor oleosin-GFP expression using real-time confocal laser scanning microscopy. We show that normally non-oil-storing tobacco leaf cells are able to synthesize and then transport oleosin-GFP fusion protein to leaf oil bodies. Synthesis and transport of oleosin-GFP fusion protein to oil bodies occurred within the first 6 h posttransformation. Oleosin-GFP fusion protein exclusively associated with the endoplasmic reticulum and was trafficked in a Golgi-independent manner at speeds approaching 0.5 microm sec(-1) along highly dynamic endoplasmic reticulum positioned over essentially static polygonal cortical endoplasmic reticulum. Our data indicate that oil body biogenesis can occur outside of the embryo and that oleosin-GFP can be used to monitor early events in oil body biogenesis in real-time.     

Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets

Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets (LD), whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum (ER). Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin-green fluorescent protein (GFP) peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein (BiP), were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.     

One-step purification of insoluble hydantoinase overproduced in Escherichia coli

Over-expression of hydantoinase from Agrobacterium radiobacter NRRL B11291 (HDTar) results in the formation of insoluble aggregates in Escherichia coli. As previously reported, recombinant HDTar could be obtained in a homogeneous form using one chromatographic step. However, soluble proteins are required for the pre-treatment in several steps before proceeding to the chromatographic purification step. In this study, we reported a method based on artificial oil bodies (AOBs) to obtain homologous HDTar from its insoluble form in one step. By linkage of HDTar to intein-oleosin gene fusion, the tripartite fusion protein was over-expressed as aggregates in E. coli. Upon sonication, the mixture comprising plant oil and the insoluble fusion protein was readily assembled into AOBs. Further induction for peptide cleavage mediated by intein, the bound HDTar was liberated from AOBs, and the protein free of fusion tags was then recovered. As a result, refolded HDTar was amplified by over 300-fold. Obviously, this simplified method provides an efficient way to obtain HDTar with high yield and high purity.     

提高Xa因子酶切效率的策略

为提高Xa因子对融合蛋白CBD-IGF和CBD-PACAP的酶切效率 ,以便高效释放非融合的重组多肽 ,利用基因工程技术在两个融合蛋白中Xa因子识别位点 (Ile-Glu-Gly-Arg↓ )前均引入 7个氨基酸组成的富含甘氨酸柔性短肽 (Gly-Thr-Gly-Gly-Gly-Ser-Gly)。纤维素亲和层析纯化各个融合蛋白 ,比较Xa因子对引入短肽前、后融合蛋白的酶切效率。比较结果表明 :短肽的引入不同程度地提高了融合蛋白CBD-IGF和CBD-PACAP对Xa因子的敏感性 ;但总体上CBD-IGF对Xa因子的敏感性比CBD-PACAP低。此研究结果提供了一种提高Xa因子酶切效率的策略。     

Stable oil bodies sheltered by a unique oleosin in lily pollen

Stable oil bodies were purified from mature lily (Lilium longiflorum Thunb.) pollen. The integrity of pollen oil bodies was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Immunodetection revealed that a major protein of 18 kDa was exclusively present in pollen oil bodies and massively accumulated in late stages of pollen maturation. According to mass spectrometric analyses, this oil body protein possessed a tryptic fragment of 13 residues matching that of a theoretical rice oleosin. A complete cDNA fragment encoding this putative oleosin was obtained by PCR cloning with primers derived from its known 13-residue sequence. Sequence analysis as well as immunological non-cross-reactivity suggests that this pollen oleosin represents a distinct class in comparison with oleosins found in seed oil bodies and tapetum. In pollen cells observed by electron microscopy, oil bodies were presumably surrounded by tubular membrane structures, and encapsulated in the vacuoles after germination. It seems that pollen oil bodies are mobilized via a different route from that of glyoxysomal mobilization of seed oil bodies after germination.     

Facilitative production of an antimicrobial peptide royalisin and its antibody via an artificial oil-body system

Royalisin found in the royal jelly of Apis mellifera is an antimicrobial peptide (AMP). It has a molecular weight of 5.5 kDa, which contains six cysteine residues. In this study, royalisin was overexpressed in Escherichia coli AD494 (DE3) as two oleosin-fusion proteins for preparation of its antibodies and functional purification. The recombinant royalisin, fused with oleosin central hydrophobic domain in both N- and C-termini, was reconstituted with triacylglycerol and phospholipids to form artificial oil bodies (AOBs). The AOBs were then purified to raise the antibodies. These antibodies could recognize both the native and recombinant royalisins, but not oleosin. Another oleosin-intein S-fusion protein was purified by AOBs system, and royalisin was subsequently released from the AOBs through self-splicing of the intein. The recombinant royalisin exhibited high antibacterial activity, which suggested that it was refolded to its functional structure. These results demonstrated that AOBs system is an efficient method to functionally express and purify small AMPs. In addition, it also provides a facile platform for the production of antibodies against small peptides.     

Expression of Human FGF18 by Fusion with Oleosin in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seeds

Expression of recombinant human fibroblast growth factor 18 (hFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, but they are difficult, expensive. The expression of recombinant proteins fused to oleosin protein have distinct advantages, such as safety, ease, low cost. So we have expressed hFGF18 fused to oleosin protein in the oil bodies of Arabidopsis thaliana (A. thaliana) and screen the proliferation effect of NIH3T3 cells. The vector of oleosinhFGF18 fusion gene was constructed and transformed into wild A. thaliana. Transformed A. thaliana lines were obtained by the floral dip method and confirmed using polymerase chain reaction (PCR). The PCR results indicated that the oleosin-hFGF18 fusion gene was integrated into the A. thaliana genome. The oil bodies expression of oleosin-hFGF18 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biological activity showed that oil bodies expressing oleosin-hFGF18 could stimulate the proliferation of NIH3T3 cells.     

Loss of XRN4 function can trigger cosuppression in a sequence-dependent manner

OLE1 encodes an oleosin isoprotein, a major membrane protein of the lipid-reserve organelle in seeds known as the oil body. Transgenic Arabidopsis were generated to contain an artificial chimeric transgene composed of OLE1 and green fluorescent protein (GFP). Overexpression of the fusion protein allowed visualization of the oil body size and structure in living cells using fluorescence microscopy. Two mutants, xrn4-8(OleG) and xrn4-9(OleG), accumulating enlarged oil bodies with reduced GFP fluorescence were isolated from the mutagenized progeny of a transgenic plant. Both mutants contained a defect in EXORIBONUCLEASE4 (XRN4), a gene known to encode a ribonuclease that specifically degrades uncapped mRNAs. Transgene expression was silenced in these mutants, as demonstrated by the reduced levels of the transgene mRNA and its product, OLE1-GFP. XRN4 loss of function also triggered cosuppression, i.e. simultaneous reduction in expression of the transgene and an endogenous OLE1 gene that shared a region of identical sequence. The enlarged oil bodies exhibiting reduced GFP fluorescence were formed in the xrn4-8(OleG) and xrn4-9(OleG) mutants due to the reduction of the endogenous OLE1 and the transgene product, OLE1-GFP, respectively. Cosuppression triggered by the xrn4 mutation also occurs for other genes such as PYK10, which encodes an endoplasmic reticulum (ER) body-resident β-glucosidase. The overall results indicate that a loss of XRN4 function can potentially trigger the cosuppression in a sequence-dependent manner.     

Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T₂ seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS-PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10–1.14 g ml^?1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studies of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bodies in vivo and of possible associated conformational/ processing changes in the protein. Although oil-body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil-body size in desiccation-tolerant seeds.     

Production of active single-chain antibodies in seeds using trimeric polyoleosin fusion

A variety of single-chain variable fragments (scFv) that had been previously developed to the surface epitopes of infective Trichostrongylus colubriformis L3 pathogenic gut nematodes of sheep were fused to a trimeric version of polyoleosin (three head-to-tail repeats of oleosin) and expressed in planta under the control of an Arabidopsis oleosin promoter. The fusion products were found to accumulate in oil bodies (OBs) at the range of 0.25-0.9% of the total seed protein which is comparable with the main 18kDa isoform of Arabidopsis seed oleosin. Immunofluorescence microscopy and immuno-binding were used to demonstrate that it is possible to both purify the recombinant protein via enrichment for OBs as well as use the OBs emulsion to deliver functional recombinant scFv. This work presents a novel fusion strategy platform to boost the productivity and simplify the delivery of recombinant single chain antibodies and other like proteins.     

A unique caleosin in oil bodies of lily pollen

In view of the recent isolation of stable oil bodies as well as a unique oleosin from lily pollen, this study examined whether other minor proteins were present in this lipid-storage organelle. Immunological cross-recognition using antibodies against three minor oil-body proteins from sesame suggested that a putative caleosin was specifically detected in the oil-body fraction of pollen extract. A cDNA fragment encoding this putative pollen caleosin, obtained by PCR cloning, was confirmed by immunodetection and MALDI-MS analyses of the recombinant protein over-expressed in Escherichia coli and the native form. Caleosin in lily pollen oil bodies seemed to be a unique isoform distinct from that in lily seed oil bodies.     

Analysis of the Three Essential Constituents of Oil Bodies in Developing Sesame Seeds

Oil bodies of plant seeds contain a matrix of triacylglycerolssurrounded by a monolayer of phospholipids embedded with alkalineproteins termed oleosins. Triacylglycerols and two oleosin isoformsof 17 and 15 kDa were exclusively accumulated in oil bodiesof developing sesame seeds. During seed development, 17 kDaoleosin emerged later than 15 kDa oleosin, but it was subsequentlyfound to be the most abundant protein in mature oil bodies.Phosphotidylcholine, the major phospholipid in oil bodies, wasamassed in microsomes during the formation of oil bodies. Priorto the formation of these oil bodies, a few oil droplets ofsmaller size were observed both in vivo and in vitro. Theseoil droplets were unstable, presumably due to the lack of sterichindrance shielded by the oleosins. The temporary maintenanceof these droplets as small entities seemed to be achieved byphospholipids, presumably wrapped in ER. Oil bodies assembledin late developing stages possessed a higher ratio of oleosin17 kDa over oleosin 15 kDa and were utilized earlier duringgermination. It seems that the proportion of oleosin 17 kDaon the surface of oil bodies is related to the priority of theirutilization. (Received July 16, 1997; Accepted October 27, 1997)     

Production of biologically active hirudin in plant seeds using oleosin partitioning

A plant oleosin was used as a carrier for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti-thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin carrier. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide.