PA2783 (Mep72) carries proteolytic and endopeptidase activities. (A) Detection of protease activity produced by PA2783 in E. coli. DH5α/pUCP19 (vector control) and DH5α/pAB2 (carrying PA2783 [mep72] under the lac promoter) were grown in LB broth and cells were spotted onto skim milk agar plates, incubated at 37°C for 48 h, and observed for zones of clearing. (B) Production of recombinant Mep72 (rMep72) in E. coli. LMG194/pAB4 (in which mep72 is expressed from the arabinose promoter) was grown in RM minimal medium supplemented with glucose overnight and subcultured into fresh RM minimal medium. At an OD600 of 0.5, 0.002% arabinose was added to induce expression of mep72 and incubation continued for 5 h. Cells were harvested, lysed, and 10 μg of whole cell lysates were separated by 10% SDS-PAGE, and stained with Coomassie blue. S, molecular mass standards; U, uninduced cells; I, induced cells; arrowhead indicates rMep72. (C) Recombinant Mep72 is detected within the outer membrane fraction of E. coli. LMG/194/pAB4 was grown as in (B). Cells were harvested and outer membranes were extracted, separated by 10% SDS-PAGE, and stained with Coomassie blue. S, molecular mass standards; U, uninduced cells; I, induced cells; arrowhead indicates rMep72. (D) Endopeptidase activity produced by rMep72 was determined as previously described. One unit equals the amount of enzyme sufficient to produce an increase in A520 of 0.001 per min at 37°C and pH 7.5 (Methods). Values represent the means of three independent experiments ± SEM. U, uninduced cells; I, induced cells.
Balyimez et al. BMC Microbiology 2013 13:269 doi:10.1186/1471-2180-13-269