Figure 1
Construction and characterization of GALA-His-Z HER2 -BNC. (A) Schematic representations of constructed His-ZHER2-BNC and GALA-His-ZHER2-BNC. The plasmid for expression of the GALA-fused His6-ZHER2-BNC (GALA-His-ZHER2-BNC) was constructed by replacing the His-ZHER2 fragment with a GALA-His-ZHER2 fragment. Briefly, a DNA fragment encoding the GALA was prepared by annealing A1 and A2 primer pairs (A1; 5'- GGG GGA TCC TGG GAA GCT GCT TTG GCT GAA GCT TTG GCT GAA GCT TTG GCT GAA CAC TTG GCT GAA GCT TTG GCT GAA GCT TTG GAA GCT TTG GCT GCT -3' and A2; 5'- GTG GTG GTG AGC AGC CAA AGC TTC CAA AGC TTC AGC CAA AGC TTC AGC CAA GTG TTC AGC CAA AGC TTC AGC CAA AGC TTC AGC CAA AGC AGC TTC CCA -3'). A DNA fragment encoding the His-ZHER2 was amplified by PCR with B1 and B2 primer pairs (B1; 5'- TTT GGC TGC TCA CCA CCA CCA CCA CCA CGC GCA ACA CG AT -3' and B2; 5'- GGG GCG GCC GCC TTT CGG CGC CTG AGC ATC AT -3') from pGLDsLd50-His-ZHER2[16]. A DNA fragment encoding the entire GALA-His-ZHER2 was amplified by an overlap PCR of the amplified fragments with A1 and B2 primer pairs, and was digested with BamH I/Not I and ligated into the same sites of pGLDsLd50-His-ZHER2. The resultant plasmid was designated as a pGLDsLd50-GALA-His-ZHER2. (B) Western blotting analyses of GALA-His-ZHER2-BNC. Sample was subjected to SDS-PAGE followed by immune blotting using anti-His6 antibody (for His6 tag; left image) and anti-protein A antibody (for ZHER2 affibody; right image). (C) Size distribution using DLS analysis. The average size of the GALA-His-ZHER2-BNC was 105Â nm. (D) Scanning electron microscope image of GALA-His-ZHER2-BNC. GALA-His-ZHER2-BNC freeze-dried with sucrose was analyzed using a JSM-7500Â F (JEOL, Munchen, Germany), following the manufacturer's procedure. Scale bar: 100Â nm.