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

We have used a strategy of hybrid gene synthesis and constant domain s huffling to construct and functionally express in Escherichia coli gen es encoding two anti-carbohydrate Fabs, one specific for a Brucella ce ll-surface polysaccharide and the second for the human blood group A d eterminant. Very similar V(L) amino acid sequences made possible the s imultaneous synthesis of the two corresponding genes. A class switchin g approach was used in Fd and light chain gene assembly. The two indep endently synthesized V(H) genes were fused to a previously made sequen ce encoding the C(gamma1)1 domain as an alternative to synthesis of th e natural C(gamma2b)1 and C(mu)1 sequences. The V(L) genes were initia lly coupled to a synthetic C(kappa) gene. When these light chain and t he above Fd genes, each preceded by the ompA signal sequence, were exp ressed from two-cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Repla cement 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 g roup 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 th e elbow region was essential for full activity of the hybrid light cha in constructs, since a two residue increase in elbow length abolished antigen binding activity. The Brucella antibody tolerated the longer e lbow sequence. Sequences in the C(lambda1) domain may result in increa sed yields of functional light chain by improving translocation across the cytoplasmic membrane or by reducing formation of periplasmic incl usion bodies.