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Tetrameric β를 이용한 고초균 포자에서의 미생물 표면 발현 모체 선별
김준형,반재구,김병기,Kim, June-Hyung,Pan, Jae-Gu,Kim, Byung-Gee 한국생물공학회 2011 KSBB Journal Vol.26 No.3
Using tetrameric ${\beta}$-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell ${\beta}$-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. ${\beta}$-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell ${\beta}$-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell ${\beta}$-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7% decrease in protease activity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.
고초균 포자를 이용한 Zymomonas mobilis 유래의 levansucrase 표면 발현
김준형(June-Hyung Kim),최수근(Soo-Keun Choi),정흥채(Heung-Chae Jung),반재구(Jae-Gu Pan),김병기(Byung-Gee Kim) 한국생물공학회 2011 KSBB Journal Vol.26 No.3
Using Bacillus subtilis spore display system, with cotG as an anchoring motif, levansucrase from Zymomonas mobilis, was displayed on the outer surface of Bacillus subtilis spore. Flow cytometry of DB104 (pSDJH-cotG-levU) spore, proved the surface localization of CotG-LevU fusion protein on the spore compared to that of DB104. Enzymatic activity of DB104 (pSDJH-cotG-levU) spore showed more than 1.5 times higher levansucrase specific activity compared to that of the host spore, which is a remarkable increase of enzymatic activity considering the existence of sacA (sucrase) and sacB (levansucrase) in the Bacillus subtilis chromosome. The spore integrity, revealed by sporulation frequency test after heat and lysozyme treatment of spore, did not changed at all in spite of the CotG-LevU fusion protein incorporation into the spore coat layer during spore formation process. These data prove again that Bacillus subtilis spore could be considered as good live immobilization vehicle for efficient bioconversion process.
Tetrameric β-galactosidase를 이용한 고초균 포자에서의 미생물 표면 발현 모체 선별
김준형(June-Hyung Kim),반재구(Jae-Gu Pan),김병기(Byung-Gee Kim) 한국생물공학회 2011 KSBB Journal Vol.26 No.3
Using tetrameric β-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell β-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. β-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell β-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell β-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7 % decrease in protease ctivity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.
Development of screening method for highly enriched peptides toward a multiple LPS using epoxy bead
Yun-Gon Kim(김윤곤),Chang-Soo Lee(이창수),Woo-Jae Chung(정우재),Eun-Mi Kim(김은미),Dong-Sik Shin(신동식),June-Hyung Kim(김준형),Yoon-Sik Lee(이윤식),Junho Chung(정준호),Byung-Gee Kim(김병기) 한국생물공학회 2005 한국생물공학회 학술대회 Vol.2005 No.10
남기연,김준형,조성준,이현용,Nam Gi-Yeon,Kim Jun-Hyung,Cho Sung-June,Lee Hyun-Yong 한국전기전자재료학회 2006 전기전자재료학회논문지 Vol.19 No.5
In this paper, we report the changes of morphology, transmittance and photoluminescence (PL) in hydrogenated amorphous $As_{40}Ge_{10}Se_{15}S_{35}$ thin films, thermally deposited at the vapor incidence angles (${\theta}$) of $0^{\circ},\;45^{\circ}\;and\;80^{\circ}$. The hydrogenation was carried out under the condition of a $H_2$ pressure ($P_H$) of 20 atm and an annealing temperature range, $T_{Anneal}$ of $150^{\circ}C{\sim}210^{\circ}C$. A columnar structures with an inclination angle of approximately $65{\sim}70^{\circ}$ was formed in $80^{\circ}$-deposited films and then the columnar was broken after hydrogenation. Transmittance increases with an increase of deposition angle and by the hydrogenation. In particular, a broad PL band on the extended region is observed in obliquely deposited films and it increases during the hydrogenation.