1. Protein induction method

1. 1. Protein induction method • One colony inoculated in 3 ㎖ LB (add ampicillin) medium and incubated in a shaking incubator overnight. • 5 ㎖ incubated cell was inoculated in 500 ㎖ LB (add ampicillin) medium and incubated in a shaking incubator of 37℃ for 3 hours. • After 3 hours, 20 ㎖ LB (add ampicillin) medium was harvested. • Add 240 ㎕0.5mMIPTG in LB (add ampicillin) medium and incubated in a shaking incubator of 30 ℃ for 3 hours. • After another 3 hours, 20 ㎖ LB (add ampicillin) medium was harvested. • Cells were Incubated every one hour (until 6 hours), 20 ㎖ medium was harvested except 4 and 5 hours ( 90 ㎖ harvested ). • Each of harvested cells were suspended in 3 ㎖ 50 mM potassium phosphate 0.5M NaCl [ pH 8.0 ] buffer. • Broke the cells with a sonicator. • Centrifugation at 15000 x g for 30 min at 4℃. • Proceeded protein assay after crude extract gained. • SDS PAGE.

2. Lane 1: Protein marker ( kDa ) Lane 2 : Cultivate at 37℃ for 3 hours ( pProEX-HTa vector) 20.2 ㎕ + SDS dye 4 ㎕ Lane 3 : Cultivate at 37℃ for 3 hours 19.7 ㎕ + SDS dye 4 ㎕ + D.W. 0.3 ㎕ Lane 4 : Add 240 ㎕ IPTG and Cultivate at 30℃ for 3 hours →18.5 ㎕ + SDS dye 4 ㎕ + D.W. 1.5 ㎕ Lane 5 : Add 240 ㎕ IPTG and Cultivate at 30℃ for 4 hours →16.9 ㎕ + SDS dye 4 ㎕ + D.W. 3.1 ㎕ Lane 6 : Add 240 ㎕ IPTG and Cultivate at 30℃ for 5 hours →17.0 ㎕ + SDS dye 4 ㎕ + D.W. 3.0 ㎕ Lane 7 : Add 240 ㎕ IPTG and Cultivate at 30℃ for 6 hours →18.5 ㎕ + SDS dye 4 ㎕ + D.W. 1.5 ㎕ 2. CbbR Protein induction ( Protein : 10 ㎍ ) 1 2 3 4 5 6 7 187 127 80 52 42 27 20 14 10 Protein size : 33 kDa