Case 2: Preparation of a complex of a transmembrane transporter and its ancillary protein, develop antibodies with therapeutic potential and resolve the 3D structure of the transporter/ancillary protein complex.
Requirements:
1.From the synthesis of transporter’s gene to its overexpression and purification.
2.Generating monoclonal antibodies with therapeutic potential potential. The antibodies are obtained from mice immunization.
3.Determining the cryo-EM structure of the transporter/ancillary protein/antibody complex.
process：
| 2 weeks | 4 weeks | 2 weeks | 1 week | 2-3 months | Time varies with different methods | 8 weeks |
| Generation of expression constructs | Preparation of baculovirus | Overexpression of antigens in HEK293 cells | Antigen purification | Mice immunization | Antibody screening | Structural analysis of antigen/antibody complex by electron microscopy |
| Design and synthesis of optimized expression vectors using bioinformatics | Obtain P0, P1, and P2 viruses | Collection of diffraction data from single crystals with sizes ranging from 1-100 um3 using high-brightness synchrotron X-ray sources | Purification of antigen protein using affinity and size exclusion chromatographies | Choosing the appropriate immunization strategy based on the target: purified protein (with detergent, liposome or nanodisc), or cells overexpressing the target protein | Hybridoma, phage display, and single B cell screening | Includes negative staining, cryo-sample preparation, data collection, 2D classification, and 3D model reconstruction |
Preparation of transmembrane transporter/ancillary protein complex and validation of the complex’s transport activity.
Production of antibodies and validation of their affinities to the complex,as well as testing their inhibitory activities at molecular and cellular levels and in mice models.
Analyzing the structure of the complex of the SLC X/ancillary protein and its antibody using single-particle cryo-EM technique.
