Article

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

ABSTRACT

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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    • "lipolytica shuttle vector pYLEX1 (Yeastern Biotech Co. Ltd.) was utilized for the oleosin–OPH fusion construction. The plasmids pINCOP [20] and pET-Ols-Xa-GFP [17] containing the OPH, and oleosin genes, respectively, were used as templates for PCR amplification. "
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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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    • "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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