Effect of C lambda-C kappa domain switching on Fab activity and yield in Escherichia coli: synthesis and expression of genes encoding two anti-carbohydrate 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 VL 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 VH genes were fused to a previously made sequence encoding the C(gamma 1)1 domain as an alternative to synthesis of the natural C gamma 2b 1 and C mu 1 sequences. The VL 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 lambda 1 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 lambda 1 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.(ABSTRACT TRUNCATED AT 250 WORDS)
MacKenzie
C R
CR
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Sharma
V
V
Brummell
D
D
Bilous
D
D
Dubuc
G
G
Sadowska
J
J
Young
N M
NM
Bundle
D R
DR
Narang
S A
SA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002002:Brucella; D002241:Carbohydrates / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005815:Genetic Code; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
D002241:Carbohydrates; D007140:Immunoglobulin Fab Fragments