3.1. Construction and expression of HER2 ECD
The gene encoding the human HER2 ECD (1968 bp in length) was amplified from cDNA synthesized on mRNA template. The PCR product was cloned into pcDNA3-SV5-BAP and pcDNA3-hεCH4-GPI vectors, downstream of the secretory signal sequences. Both recombinant vectors were verified by digestion and agarose gel size. Results of sequence analysis confirmed the sequences of following proteins HER2 ECD-SV5-BAP from pcDNA3 HER2 ECD-SV5-BAP, and HER2 ECD-hεCH4-GPI from pcDNA3 HER2 ECD-hεCH4-GPI recombinant vectors. For in vivo biotinylation of HER2 ECD, equimolar amounts of both plasmids pcDNA3 HER2 ECD-SV5-BAP and pCDNA3-BirA encoding the tagged HER2 ECD and ...view middle of the document...
2-C). The results of Western blotting showed that most molecules of HER2 ECD were biotinylated and hence the efficiency of the method was high.
3.2. Purification and characterization of recombinant protein
Culture supernatants from selected clone was harvested and processed for purification of biotinylated HER2 ECD using Avidin Resin. Protein purification was analyzed by SDS–PAGE that stained with Coomassie blue. Figure 2-D shows the high purity of protein. In Bradford assay the amount of purified protein was estimated to 15 mg/l. Finally Western blotting analysis with anti-HER2 mAb confirmed the identity of biotinylated HER2 ECD (Fig. 2-E)
The binding of two anti HER2 scFvs, scFvHL-8 and scFvHL-10, to recombinant HER2 ECD was proved by a Precipitation assay. The scFv proteins were incubated with culture supernatant of the selected cells expressing the biotinylated protein. Subsequently streptavidin magnetic beads were used to precipitation and separation of bound proteins to ECD of HER2. Finally, the captured bead-protein complexes were boiled and run on an SDS-PAGE gel. The presence of protein bands, scFvHL-8, scFvHL-10, and HER2 ECD was detected in Western blotting analysis by anti-His (epitop tag of scFvs) and anti-SV-5 antibodies, respectively. As shown in figure 2-F, both scFvs were able to recognize the extracellular domain of HER2 specifically.
3.3. Analysis of the transfectant HER2 ECD-GPI anchored cells
A recombinant anchored version of the HER2 ECD protein was also expressed in its membrane bound form. SP2/0 cells were stably transfected with pcDNA3 HER2 ECD-hεCH4-GPI construct. Single cell-derived clones were isolated from stable transfectants by Genticin selection and expanded to analyze by Immunoblotting, FACS and Immunoflourescent microscopy assays.
Cell lysates of two selected clones showed the expression of εCH4- tagged HER2 ECD in Western blot analysis (Fig. 3-A). The recombinant HER2 ECD with εCH4 and GPI anchored had a molecular mass around 90 kDa that was detected with anti εCH4 mAb. Following the Western blot analysis, the ability of anti HER2 mAb for detection of cells expressing HER2 ECD-GPI was demonstrated in FACS assay. Flow cytometric analysis showed that clone 2 expressed HER2 ECD-GPI anchored protein significantly more in comparison with clone 1 (FIG. 3-B). Furthermore, the expression of the membrane bound version of the HER2 ECD in clone 2 cells was determined by fluorescence microscopy. Recombinant protein was detected by using an anti-εCH4 antibody. As revealed by immunofluorescent microscopy, the membrane form of HER2 ECD was effectively displayed on the stable transfected cell surface (Fig. 3-D).
The extracellular domain of HER2 protein was expressed and purified in this study. Biotinilation can exploit, not only for purification of HER2 ECD but also for protein targeting and isolation of binders that can interact with this protein in a selection assay . Biotin has a strong interaction...