Figure 2.

Western blot of single-chain variants expressed with or without a leader peptide. (A) Variant NEGM was fused to different leader peptides or expressed without a leader peptide. (B) The three indicated variants were expressed without a leader peptide or (C) with a modified β-lactamase leader peptide. The PGA wild-type protein was expressed using its natural leader peptide. All the cultures were OD₆₀₀ normalized. T means a total protein sample, and S means a soluble protein sample. The upper band in the PGA-WT lane corresponds to the unprocessed polypeptide (precursor protein) and the lower band corresponds to the β-subunit. The percentages indicated below the soluble protein bands in parts B and C correspond to the relative specific activities measured using 20 times the protein amount shown in the figure; 11.66 μg per min of produced 6-APA using 100 μg of soluble protein of the wild-type clone was taken as 100%.