Cloning and expression of an autoimmune DNA-binding single chain Fv. Only the heavy chain is required for binding.
Hed 10 is a murine autoimmune antibody which binds tightly to the single-stranded DNA, poly(dT). The heavy and light chain variable region genes of Hed 10 were cloned and then joined by a 42 base-pair linker with the aid of a PCR ligation technique to produce a single chain Fv gene. After insertion into an expression vector the single chain Fv protein (scFv Hed 10) was produced in high yield and was purified by chromatography on an oligo (dT) cellulose column. The binding of scFv Hed 10 to poly (dT) was measured by fluorescence quenching and a binding constant of 3.2 x 10(6) M-1 was calculated. Previously [Lee et al. (1982) Biochemistry 21, 4940-4945] the binding constant of Fab Hed 10 to poly (dT) was found to be 12.7 x 10(6) M-1. In addition the Vh gene of Hed 10 was expressed independently as well as another scFv which contained the Vh region of Hed 10 linked to the light chain variable region of Jel 42, an antibody specific for Hpr protein of E. coli (scFv 10 H.42 L). Both of these proteins had binding constants for poly (dT) in the range of 6 x 10(6) M-1. Therefore, the light chain of Hed 10 contributes little to the binding of this autoimmune antibody to DNA.
Barry
M M
MM
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Lee
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D001323:Autoantibodies / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011067:Poly dA-dT / Q000378:metabolism
D001323:Autoantibodies; D004268:DNA-Binding Proteins; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv; D011067:Poly dA-dT