Fig. 1
A comparison of commonly used signal peptides. a An overview of the expression cassettes used in this experiment. The TIR is represented by the boxed area, which extends from the Shine-Dalgarno to the fifth codon of the signal sequence. The coding sequences (CDS) for five commonly used signal peptides (MalESP, OmpASP, PhoASP, DsbASP, PelBSP) were cloned into the pET28a vector, upstream of the mature CDS for β-lactamase, scFvHER2 or FtYfgM45−170. Protein production was induced for 2 h, then a volume of cells corresponding to 0.2 OD600 units of cells were harvested, separated by a 12% SDS-PAGE and protein levels determined by immuno-blotting with antisera to β-lactamase b, or the poly-Histidine tag of scFvHER2c and FtYfgM45–170d. To ensure that protein loading was consistent between the samples, the membranes were stained with Amido black after immuno-detection. ‘Pre’ denotes the precursor form of the protein, which contains the signal sequence and is presumed to be in the cytoplasm. ‘Mat’ precursor denotes the mature form, which is presumed to be in the periplasm as the signal peptide has been cleaved