Tof tof tm 5800

The SCP‐tagged BPTI variants were constructed by introducing the SCP‐tags to either the N or C termini of BPTI‐19A, a simplified BPTI variant [22] containing 19 alanines out of its 58 residues [23] (Fig. 1). BPTI variants were named according to the number and type of amino acids added to the N or C terminus of BPTI‐19A. The DNA sequences encoding the SCP‐tags were added by QuikChange site‐directed mutagenesis (Stratagene, California, USA) using pMMHa BPTI‐19A vector as a template [23], and the plasmid sequences were confirmed by DNA sequencing. The tagged variants were expressed in Escherichia coli BL21 (DE3) pLysS cell lines as inclusion bodies and were solubilized in 6 M GuHCl with overnight oxidation at 25 °C. CNBr (cyanogen bromide) reaction was then performed in order to cleave the TrpΔLE leader sequence, and proteins were purified by pI precipitation followed by reverse phase HPLC [18]. Furthermore, protein identities were confirmed by MALDI‐TOF mass spectrometry (AB SCIEX TOF/TOF TM 5800, Framingham, USA), and the purified proteins were preserved at −30 °C as lyophilized powders. A schematic representation of protein purification is given in Fig. S4.