Figure 2. Heterologous expression of modified PedA15 peptides in E. coli and analysis by MALDI-TOF MS.

(A) Coexpression in E. coli BL21 (DE3) of PedA15 precursor peptides, PedB15 and PedC15 in the absence of P. lusitanus tRNA^Glu/GluRS. Both PedA15.1 (left; avg. m/z 8687.7 calc.; 8686.6 obs.) and PedA15.2 (right; avg. m/z 8085.0 calc.; 8085.4 obs.) were unmodified in the absence of aminoacyl-tRNA from the native producer. Consequently, NEM treatment of these samples resulted in full alkylation of all Cys residues (grey spectra). (B) Comparison of tRNA^Glu sequences in E. coli (Proteobacterium) and P. lusitanus NL19 (Bacteroidetes). The anticodon is denoted in subscript. Nucleotide positions previously shown to be important for LanB compatibility are in bold.¹⁵ (C) PedA15 precursor peptides co-expressed with PedB15, PedC15 and P. lusitanus tRNA^Glu/GluRS. Isolated product for PedA15.1 (left) contained a mixture of 4x (avg. m/z 8615.7 calc.; 8615.8 obs.) and 5x (avg. m/z 8597.6 calc.; 8597.0 obs.) dehydrated peptide and was fully cyclized as evidenced by a lack of NEM alkylation. PedA15.2 (right) was purified as a mixture of 3x (avg. m/z 8030.9 calc.; 8030.5 obs.) and 4x dehydrated (avg. m/z 8012.9 calc.; 8012.7 obs.) peptide, with a minor amount of partially cyclized peptide product observed by NEM alkylation. M, parental mass (avg. m/z, unmodified peptide); NEM, N-ethylmaleimide (grey traces); “-n” H₂O, indicates the number (n) of dehydrations consistent with the m/z of the labeled peak.