A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides.
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.
Peng
Chi-Chung
CC
Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan.
Chen
Jeff C F
JC
Shyu
Douglas J H
DJ
Chen
Ming-Jinn
MJ
Tzen
Jason T C
JT
D002498:Centrifugation / Q000379:methods; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D015951:Factor Xa / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*
D029968:Escherichia coli Proteins; D010455:Peptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C110161:oleosin protein, Sesamum indicum; D015951:Factor Xa