Chun-Xia Zhao
1
, Mirjana Dimitrijev Dwyer
1
, Alice Lei Yu, Yang Wu, Sheng Fang, Anton P. J.
Middelberg*
The University of Queensland, Australian Institute for Bioengineering and Nanotechnology, St.
Lucia QLD 4072, Australia
*Corresponding author; Email: a.middelberg@uq.edu.au; Tel: +61 7 3346 4189; Fax: +61 7
3346 4197.

Expression screen of DAMP4-omiganan (MDPS MKQLADS LHQLARQ VSRLEHA DPS
MKQLADS LHQLARQ VSRLEHA DPS MKQLADS LHQLARQ VSRLEHA DPS MKQLADS
LHQLARQ VSRLEHA DENLYFQ GILRWPWWPWRRK HHHHHH)
DNA encoding the omiganan peptide was annealed and cloned downstream of DAMP4 using standard molecular biology techniques. The construct was transformed into E.coli
BL21(DE3).
For each construct, a starter culture was grown from a single colony picked from transformation plates (LB agar - Kan 30 μg/mL). This starter culture was used to inoculate duplicate cultures of
105 mL of media (2xYT - Kan 15 μg/mL) in 250 mL shake flasks. Both cultures were incubated at 37°C 180 rpm until the OD
600
reached 0.5, at this point one culture was cooled to room temperature before induction with 1 mM IPTG and further incubated at 25°C, while the other culture was induced immediately with 1 mM IPTG and further incubated at 37°C. All cultures were incubated overnight (O/N) until harvest (22 h post induction).
Analysis
30 mL samples were taken at 0 h, 4 h, 7 h, and O/N time-points for expression and solubility analysis. The 30 mL harvest samples were spun down and resuspended in 30 mL of lysis buffer
(25 mM sodium phosphate, 50 mM sodium chloride, 0.5% Triton X-100, 2 mM magnesium chloride pH 7.5) and lysed by sonication. All samples were analysed by SDS-PAGE for expression and solubility, as shown in Figure S1.
Figure S1 Expression and solubility analysis of DAMP4-omiganan. (Lane 1) Ladder, (lane 2) 0 h total protein, (lane 3) 0 h soluble protein, (lane 4) 4 h, 25°C, total protein, (lane 5) 4 h, 25°C, soluble protein, (lane 6) 4 h, 37°C, total protein, (lane 7) 4 h, 37°C, soluble protein, (lane 8) 7 h,
25°C, total protein, (lane 9) 7 h, 25°C, soluble protein, (lane 10) 7 h, 37°C, total protein, (lane
11) 4 h, 37°C, soluble protein, (lane 12) O/N, 25°C, total protein, (lane 13) O/N, 25°C, soluble protein, (lane 14) O/N, 37°C, total protein, (lane 15) O/N, 37°C, soluble protein.