Abstract
The antigen receptor of B-lymphocytes (BCR) plays important roles in the recognition of foreign antigens and in the generation of appropriate antibody responses. The BCR is composed of membrane-bound immunoglobulin (mIg) and two pairs of Ig-αand Ig-βdimers. Ig-αor Ig-βcontains an immunoglobulin-fold structure in the extracellular part, a transmembrane region, and a cytoplasmic tail,which is longer than those in mIg. The extracellular domains of Ig-αand Ig-βhave not been produced in recombinant forms. To elucidate the roles of Ig-αand Ig-βand their relationship with mIg in BCR, we prepared the extracellular domains of human Ig-αand Ig-βby expressing them in Escherichia coli. The following steps were performed: (1) the genes of human Ig-αand Ig-β were cloned; (2) appropriate expression vectors (GST, pQE, MBP gene fusion vectors) were chosen and recombinant plasmids containing Ig-αand Ig-βgenes were constructed; (3) optimal conditions (E.coli stains, incubation time, induction temperature and inducer concentration) for the expression of soluble Ig-αand Ig-βfusion proteins were explored by detecting expressed products in reduced SDS-PAGE; (4) the fusion proteins were expressed and purified with affinity chromatography. Among the selected expression systems, only MBP fusion system expressed desired proteins in soluble form. The affinity-purified MBP human Ig-αand Ig-βfusion proteins exhibited the expected sizes of Mr 56 kDa and 58 kDa, respectively on reduced SDS-PAGE. The extracellular domains of human Ig-αand Ig-βwere cleaved from the fusion proteins by digestion with factor Xa . These purified proteins will be used for the production of monoclonal antibodies against human Ig-αand Ig-β and provide as useful reagents for structural analysis on the interaction between Ig-αand Ig-βwith mIg.