Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity.
Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of beta2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasabeta2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens.
Zhao
Heng
H
Department of Food Science and Nutritional Engineering, China Agricultural University, 100083, Beijing, PR China. hengzhao2000@gmail.com
Stet
René J M
RJ
Skjødt
Karsten
K
Savelkoul
Huub F J
HF
D000818:Animals; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D015870:Gene Expression / Q000235:genetics*; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020317:Salmo salar / Q000235:genetics / Q000378:metabolism*; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015395:Histocompatibility Antigens Class I; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin