Korean Society for Biotechnology and Bioengineering Journal Korean Society for Biotechnology and Bioengineering Journal

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Korean Society for Biotechnology and Bioengineering Journal 2011; 26(3): 199-205

Published online June 30, 2011

Copyright © Korean Society for Biotechnology and Bioengineering.

Tetrameric β를 이용한 고초균 포자에서의 미생물 표면 발현 모체 선별

Screening of Bacterial Surface Display Anchoring Motif Using Tetrameric β-galactosidase in Bacillus subtilis Spore

김준형(동아대학교 화학공학과), 반재구(한국생명공학연구원), 김병기(서울대학교 화학생물공학부)

Abstract

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.

Keywords: surface display, Bacillus subtilis, spore, ${\beta}$-galactosidase