Trastuzumab-LLO Conjugation Protocol

OPSS-PEG-NHS (molecular weight 5000) was purchased from Nanocs (New York, NY, USA). Trastuzumab (Herceptin) was a generous gift from Dr Virginia Borges (UC Denver, Denver, CO, USA). LLO from Escherichia coli transfected with the LLO-pEt29-DP-E3570 plasmid, kindly provided by Dr Dan Portnoy (UC Berkeley, Berkeley, CA, USA), was purified by the method described previously^{13 (link), 14 (link)} and stored in storage buffer (50 mM phosphate buffer, pH 6.0, 1 M NaCl and 1 mM EDTA) without dithiothreitol to preserve its activity. Polylysine hydrobromide (molecular weight 37 000; degree of polymerization: 177) and 2-iminothiolane-HCl (Traut's reagent) were purchased from Sigma Life Science (St Louis, MO, USA). CL-4B Sepharose used for the purification of the one-component complexes was purchased from Amersham Biosciences (Uppsala, Sweden). All other reagents, unless otherwise specified, were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA).