Figure 1

Expression of pknbp gene segments in COS7 cells and binding profile to rhesus erythrocytes. (A) Three regions spanning the 5′ end of the coding region of PkNBPXa were cloned into pDisplay as overlapping 500 nucleotide gene segments. The numbers listed correspond to the amino acid positions delineating a particular expressed gene segment. Though the recombinant proteins were expressed intracellularly, they never reached the surface of the COS7 cells (data not shown). Eight recombinant segments (Regions I - VIII) spanning the N-terminal half of PkNBPXb were cloned and expressed in COS7 cells. (B) Protein expression for each COS7/PkNBPXb region was evaluated by immunoblot using a specific antibody against the c-myc tag encoded in the C-terminus of the pDisplay vector. (C) Rhesus monkey erythrocytes were found to adhere only to PkNBPXb-II forming a large rosette pattern on the surface of COS7 cells. Panels 1 and 2 show images representing nuclear staining with Hoechst dye, COS7 cell protein expression detected by anti-HA and Alexa Fluor 488-labeled anti-mouse IgG, and erythrocyte rosetting assay results, respectively. Top and bottom panels show the surface expression and binding capability of Region II and Region VIII, respectively. Region VIII showed high expression by immunoblot while displaying no binding affinity to rhesus erythrocytes. Images were captured at room temperature using a Nikon ECLIPSE TE 300 inverted microscope using the Plan Fluor ELWD 40x / 0.45 aperture and 10x magnification eye piece and analysed using the Diagnostic Instrument, Inc. software.