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Display of native proteins on Bacillus subtilis spores.
Pan, Jae-Gu,Choi, Soo-Keun,Jung, Heung-Chae,Kim, Eui-Joong Published by Elsevier/North Holland on behalf of t 2014 FEMS microbiology letters Vol.358 No.2
<P>In principle, protein display is enabled by fusing target proteins to naturally secreted, surface-anchored protein motifs. In this work, we developed a method of native protein display on the Bacillus spore surface that obviates the need to construct fusion proteins to display a motif. Spore coat proteins are expressed in the mother cell compartment and are subsequently assembled and deposited on the surface of spores. Therefore, target proteins overexpressed in the mother cell compartment during the late sporulation phase were expected to be targeted and displayed on the spore surface. As a proof of principle, we demonstrated the display of carboxymethylcellulase (CMCase) in its native form on the spore surface. The target protein, CMCase, was expressed under the control of the cry1Aa promoter, which is controlled by σ(E) and σ(K) and is expressed in the mother cell compartment. The correct display was confirmed using enzyme activity assays, flow cytometry, and immunogold electron microscopy. In addition, we demonstrated the display of a β-galactosidase tetramer and confirmed its correct display using enzyme activity assays and protein characterization. This native protein display system, combined with the robust nature of Bacillus spores, will broaden the range of displayable target proteins. Consequently, the applications of display technology will be expanded, including high-throughput screening, vaccines, biosensors, biocatalysis, bioremediation, and other innovative bioprocesses.</P>
임성근,안태호,정흥채,Jae-Gu Pan,윤철호 대한약학회 2005 Archives of Pharmacal Research Vol.28 No.4
Human cytochromes P450 (CYP) 2A6 and 2E1 are of great interest because of their important roles in the oxidation of numerous drugs and carcinogens. Bacterial expression systems, especially Escherichia coli cells, have been widely used for the production of various CYP enzymes in order to obtain high yield of proteins. The expression methods usually employ longer culture time (30-72 h) at lower temperature (usually under 30oC). Expression levels of CYPs 2A6 and 2E1 at 37oC were compared to those at 28oC, which is a usual temperature used in most bacterial expression systems for human CYP expression. Within 18 h the expression levels of CYPs 2A6 and 2E1 reached up to 360 and 560 nmol per liter culture at 37oC, respectively, which are compatible with those of 36 h culture at 28oC. The activities of CYPs expressed at 37oC were also comparable to those expressed at 28oC. The present overexpression system can be useful for rapid production of large amounts of active human CYPs 2A6 and 2E1 in E. coli. Human cytochromes P450 (CYP) 2A6 and 2E1 are of great interest because of their important roles in the oxidation of numerous drugs and carcinogens. Bacterial expression systems, especially Escherichia coli cells, have been widely used for the production of various CYP enzymes in order to obtain high yield of proteins. The expression methods usually employ longer culture time (30-72 h) at lower temperature (usually under 30oC). Expression levels of CYPs 2A6 and 2E1 at 37oC were compared to those at 28oC, which is a usual temperature used in most bacterial expression systems for human CYP expression. Within 18 h the expression levels of CYPs 2A6 and 2E1 reached up to 360 and 560 nmol per liter culture at 37oC, respectively, which are compatible with those of 36 h culture at 28oC. The activities of CYPs expressed at 37oC were also comparable to those expressed at 28oC. The present overexpression system can be useful for rapid production of large amounts of active human CYPs 2A6 and 2E1 in E. coli.