bccm bccm lmbp technologiepark 927 9052 zwijnaarde belgium under deposit number lmbp 6818 on 8 nov 2010 the construction of the plt32 m2e tgcn4 expression vector was described earlier de filette et al 2008 construction of the plh36 hisdevd lpp 5rshe expression plasmid a plasmid containing the coding sequence of the lpp s tryptophan zipper fused to the coding sequence of the sh ectodomain separated by the coding sequence of a glygly linker was ordered at genscript this coding sequence was amplified using the following forward and reverse pnmers 5 u2032 gcgaaatgggatcagtggagcagc 3 u2032 seq id n 53 5 u2032 aatataggatccctaggtcgcccagttatcccagcg 3 u2032 seq id n0 54 phosphorylated and digested with bamm the plh36 hisdevd lpp she was constructed by a three point ligation using the described pcr fragment bamm pstl digested plt32 plasmid fragment and ecorv pstl digested plh36 fragment the sequence of the constructed plh36 hisdevd lpp srshe plasmid is displayed in seq id n0 49 a 30 ml preculture of plt32she tgcn4 transformed e coli was grown at 28 u00b0 c in luria broth and used to inoculate 1 liter of fresh medium at an a600 of 0 6 0 8 the cells were treated with 1 mm isopropyl 1 thio d galactopyranoside incubated for another four hours and then collected by centrifugation 6000 u00d7 g 20 minutes 4 u00b0 c the bacterial pellet was resuspended in 20 ml tris hcl buffer 50 mm tris hcl 50 mm nacl and 1 mm edta ph 8 and sonicated bacterial debris.