In view of the success in preparing a single-chain hCG of the form N-fi-a-C, we then designed a single-chain hCG-LHR, i.e., yoked hCG-LHR (Fig. 2), with a C-terminal peptide (CTP) and Factor Xa cleavage site serving as linker between a and receptor. buy ortho tri-cyclen online
The yoked hCG-LHR complex was expressed in COS-7 and HEK 293 cells as evidenced by Western blot analysis using an antibody to the hCGfi-CTP peptide. The degree of [125I]hCG-specific binding was, however, relatively low, particularly in the COS-7 cells, consistent with occupancy of the expressed LHR with covalently attached yoked hCG. Using the transfected HEK 293 cells, which contained a greater degree of specific binding than the COS-7 cells, a competitive binding curve was generated with expressed wild-type LHR serving as control (Fig. 4a). Of interest is the observation that the yoked hCG-LHR complex exhibits an apparent higher affinity for hCG than wild-type LHR. That the yoked hCG-LHR complex is functional is evidenced by the results on cAMP production shown in Figure 4b. First, it should be noted that the basal level of cAMP, i.e., in the absence of exogenous hCG, is considerably higher in cells transfected with yoked hCG-LHR than in mock-transfected cells or cells transfected with LHR. Second, the yoked hCG-LHR complex remains somewhat responsive to exogenous hCG, but the fold-ac-tivation is low compared to that of LHR.
FIG. 4. a) Competitive binding of [,2Sl]hCG and hCG to HEK 293 cells expressing yoked hCG-LHR (solid squares) and wild-type LHR (open circles), b) Cyclic AMP response to hCG in HEK 293 cells expressing yoked hCG-LHR (squares) and wild-type LHR (circles); the lack of response of mock-transfected cells is also indicated (triangles). (Data from.)