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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>
Lee, Su-Jin,Pan, Jae-Gu,Park, Seung-Hwan,Choi, Soo-Keun Elsevier 2010 Journal of biotechnology Vol.149 No.1
<P><B>Abstract</B></P><P><I>Bacillus thuringiensis</I> produces crystal proteins (Cry) that account for up to 25% of the dry cell weight during the stationary phase. The high-level expression and stationary phase-specific autoinduction of the <I>cry</I> gene led to development of a <I>cry</I> promoter-based <I>Bacillus</I> expression system. Among the various <I>cry</I> promoters, <I>cry3Aa</I> promoter was selected by comparing the <I>lacZ</I> expression levels in <I>Bacillus subtilis</I>. An extracellular enzyme cellulase was highly upregulated during the stationary phase while under control of the <I>cry3Aa</I> promoter. Improvement of the <I>cry3Aa</I> promoter was obtained by modification of the promoter sequence. Specifically, a 5-fold increase in <I>lacZ</I> expression was obtained by changing both the −35 and −10 boxes of the <I>cry3Aa</I> promoter to the consensus sequence of the σ<SUP>A</SUP>-dependent promoter of <I>B. subtilis</I>. The modified <I>cry3Aa</I> promoter produced a significantly higher yield of AprE, which suggests that the promoter may be useful for high-level protein expression in <I>B. subtilis</I>.</P>
김상구 ( Kim Sang Gu ),손희종 ( Son Hui Jong ),노재순 ( No Jae Sun ),최동훈 ( Choe Dong Hun ),배석문 ( Bae Seog Mun ),권기원 ( Kwon Gi Won ),신판세 ( Sin Pan Se ),김원경 ( Kim Won Gyeong ) 한국수처리학회 2003 한국수처리학회지 Vol.11 No.3
The performance of a novel magnetic ion exchange resin supplied by its manufacturer Orica Australia(M1EX? DOC Resin) in terms of the removal of dissolved organic carbon(DOC). reduction in disinfection by-product formation potential, and effects on the removal of turbidity has been investigated. Two water samples(Maeri and prepared raw waters) were first treated with MIEX? at various doses and contact times, followed by conventional coagulation. Measurements of DOC. UV-254. THMFP, HAAFP and turbidity were made at each stage. In addition, readings of anions, including Br- were also obtained. For comparison, conventional coagulation/sedimentation alone was carried out using fixed levels of coagulant of 60㎎/L for Maeri water, and 100 ㎎/l for prepared water respectively. The UV-254 and DOC removals by MIEX? treatment were shown to be superior to cuagulation, ie better by 11-40% and 20-26% for Maeri and prepared raw water respectively. Further coagulation of the 10 mL/L MIEX? treated waters to control turbidity increased the DOC removal by -16%. As MIEX? dose and contact time increased, the additional DOC removal by coagulation showed corresponding decreases. The DOC removal efficiencies by MIEX? treatment alone were 71% and 82% with Maeri and prepared raw waters respectively. while the corresponding figures achieved by coagulation alone were 48% and 49% respectively. The combination of MIEX? and coagulation(main1y to achieve treatment standard for turbidity) in series yielded DOC removal efficiencies of 83% and 88% for the two waters. The reduction in THMFP by MIEX? treatment was better than that achieved by coagulation by 20-49%. More significantly, MIEX? treatment delivered a reduction of HAAFP which was 50% better than that delivered by coagulation. However, the combination of MIEX? and coagulation, when compared with MIEX? treatment alone, showed only marginal improvements in THMFP and HAAFP. indicating that as a pre-treatment method, MIEX? was particularly effective in removing DBP precursors. MIEX? treatment significantly reduced the dose of coagulant which would be needed for turbidity control. In order to reduce the turbidity from 18(Maeri water) and 27 NTU(prepared raw water) to ( 2 NTU, MIEX? pre-treated water samples only required coagulant doses which were up to 80% less than those involved when coagulation alone was used. However. after MIEX? pre-treatment, low alkalinity waters(1ess than 20 ㎎/L) only showed a slight decrease in turbidity. Most anions in water decreased with MIEX? dose and contact time with Br- showing a reduction of up to 70% while Cl- showing a slight increase due to the effect of resin regeneration. This research showed that for Maeri water(5 ㎎/L DOC and 30 NTU), the pre-treatment with MIEX? dose of 10-16 mL/L and a contact time of 30 minutes yielded optimum results for DOC and DBP control. Coagulant dosage for turbidity control in this case decreased by as much as 20-30 ㎎/L(80%).