Figure 3

In vitro Pam production. (A) Western blot confirmation of the temperature-dependent secretion of Pam in P. asymbiotica ATCC43949: the protein is present in supernatants from cultures grown at 28°C but not at 37°C. (B) Western blot confirmation of the pam knock-out in P. luminescens TT01 (left) compared with the wild-type strain (right). Note that figures A and B share the same molecular markers. (C) Pam heterologous production in E. coli. The arrow shows high levels of the recombinant Pam protein from P. asymbiotica ATCC43949 produced in E. coli. (D) Pam was purified by two steps of anion-exchange chromatography and the eluted fractions were analysed by SDS-PAGE. Lane 1: Proteins from overnight culture, lanes 2-5: elution fractions from the second ion-exchange column. The estimated purity of recombinant Pam was 95%.