Part:BBa_K2622045:Experience

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Applications of BBa_K2622045

Amplified gene sequence of BamD was ligated into a plasmid pET22b. pelB signal sequence of BamE directs protein into periplasm. BamE can be purified in subcomplex with BamC and BamD using histidine tag only on BamE. To purify BamE, E. coli cells were induced with 0,1mM IPTG, grown in 37*C for 3 hours, then pelleted and sonicated, membrane fraction was isolated by centrifugation and was further purified with Ni-NTA and gelfiltration. BamE was used in a folding assay, which showed that BamB and BamCDE lipoproteins can assemble BamA into the membrane with an efficiency higher than 50% in time range of only two hours.

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