Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.
Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.
Chiang
Chung-Jen
CJ
Department of Life Science and Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan.
Chen
Hong-Chen
HC
Chao
Yun-Peng
YP
Tzen
Jason T C
JT
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D004792:Enzyme Precursors; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D047668:Inteins; D010938:Plant Oils / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000737:chemistry; D012995:Solubility; D013381:Subtilisins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D004792:Enzyme Precursors; D010938:Plant Oils; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013381:Subtilisins; C053771:nattokinase