EFFECT OF C-LAMBDA-C-KAPPA DOMAIN SWITCHING ON FAB ACTIVITY AND YIELD IN ESCHERICHIA-COLI - SYNTHESIS AND EXPRESSION OF GENES ENCODING 2 ANTICARBOHYDRATE FABS

We have used a strategy of hybrid gene synthesis and constant domain shuffling to construct and functionally express in Escherichia coli genes encoding two anti-carbohydrate Fabs, one specific for a Brucella cell-surface polysaccharide and the second for the human blood group A determinant. Very similar V(L) amino acid sequences made possible the simultaneous synthesis of the two corresponding genes. A class switching approach was used in Fd and light chain gene assembly. The two independently synthesized V(H) genes were fused to a previously made sequence encoding the C(gamma1)1 domain as an alternative to synthesis of the natural C(gamma2b)1 and C(mu)1 sequences. The V(L) genes were initially coupled to a synthetic C(kappa) gene. When these light chain and the above Fd genes, each preceded by the ompA signal sequence, were expressed from two-cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Replacement of the C(kappa) domains with a C(lambda1) domain resulted in a significant increase in the amount of soluble periplasmic light chain and functional Fab for both the Brucella and blood group A antibodies. The C(kappa) and C(lambda1) forms of each of the Brucella and blood group A Fabs, with His5 fusions at the C-termini of the Fd chains, were purified by immobilized metal affinity chromatography. For the blood group A antibody, it was shown by ELISA that precise engineering of the elbow region was essential for full activity of the hybrid light chain constructs, since a two residue increase in elbow length abolished antigen binding activity. The Brucella antibody tolerated the longer elbow sequence. Sequences in the C(lambda1) domain may result in increased yields of functional light chain by improving translocation across the cytoplasmic membrane or by reducing formation of periplasmic inclusion bodies.