A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides

Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan.
Journal of Biotechnology (Impact Factor: 2.87). 08/2004; 111(1):51-7. DOI: 10.1016/j.jbiotec.2004.03.013
Source: PubMed


An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant.

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Available from: Chi-Chung Peng, Oct 11, 2015
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    • "Whether this is the case for LDSP as well, or if it localizes to the LD merely due to its high hydrophobicity, remains to be determined. It should be pointed out that oleosins have the ability to stabilize artificial LDs (Peng et al., 2004), suggesting that they are capable of spontaneous insertion into a lipid matrix. "
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    ABSTRACT: As our understanding of the dynamics of lipid droplets (LDs) in animal, plant, and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstock for the production of biofuels and high-value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic lipid droplet surface protein (LDSP) with unique primary sequence but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount of triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis (Arabidopsis thaliana) OLEOSIN1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.
    Plant physiology 02/2012; 158(4):1562-9. DOI:10.1104/pp.111.193029 · 6.84 Impact Factor
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    • "In particular, AOBs prepared from the recombinant Ole expressed in Escherichia coli are comparable in size, topology, and stability to OBs encapsulated with native Ole derived from plant seeds [16] [18]. The practicality of preparing AOBs has ushered in many useful applications, including a bacterial expression/purification system for producing recombinant proteins [19] [20], encapsulation of probiotics for oral administration [21], and enzyme immobilization [22]. Given that they are small, stable, biocompatible, and biodegradable, AOBs (mainly comprising natural lipids) with hydrophobic cores have the potential as a delivery carrier. "
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    ABSTRACT: A novel delivery carrier was developed using artificial oil bodies (AOBs). Plant seed oil bodies (OBs) consist of a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with the storage protein oleosin (Ole). Ole consists of a central hydrophobic domain with two amphiphatic arms that extrude from the surface of OBs. In this study, a bivalent anti-HER2/neu affibody domain (ZH2) was fused with Ole at the C terminus. After overproduction in Escherichia coli, the fusion protein (Ole-ZH2) was recovered to assemble AOBs. The size of self-assembled AOBs was tailored by varying the oil/Ole-ZH2 ratio and pH to reach a nanoscale. Upon co-incubation with tumor cells, the nanoscale AOBs encapsulated with a hydrophobic fluorescence dye were selectively internalized by HER2/neu-overexpressing cells and displayed biocompatibility with the cells. In addition, the ZH2-mediated endosomal entry of AOBs occurred in a time- and AOB dose-dependent manner. The internalization efficiency was as high as 90%. The internalized AOBs disintegrated at the non-permissive pH (e.g. in acidic endosomes) and the cargo dye was released. Results of in vitro study revealed a sustained and prolonged release profile. Taken together, our findings indicate the potential of AOBs as a delivery carrier.
    Nanotechnology 01/2011; 22(1):015102. DOI:10.1088/0957-4484/22/1/015102 · 3.82 Impact Factor
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    • "Oleosin is a protein originated from plant that has been used as a carrier to produce protein of biological interest in plants [18], [19]. Our approach here in using oleosin as an intracellular anchor for secretory protein in mammalian cells is oriented from another perspective, focusing more on the targeting ability of the protein oleosin. "
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    ABSTRACT: Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin. chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells. vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration. the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest.
    PLoS ONE 02/2009; 4(7):e6325. DOI:10.1371/journal.pone.0006325 · 3.23 Impact Factor
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