title: Two-phase approach for the expression of high-affinity human anti-human immunodeficiency virus immunoglobulin Fab domains in Escherichia coli.
abstract: We describe here a two-phase approach for the development of high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4 x 10(-10) M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 with a dissociation constant on the order of 8 x 10(-10) M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage. The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Laboratory of Oral Medicine, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: S
authors.initials: S
authors.lastname: Takeda
authors.forename: N A
authors.initials: NA
authors.lastname: Dorfman
authors.forename: M
authors.initials: M
authors.lastname: Robert-Guroff
authors.forename: A L
authors.initials: AL
authors.lastname: Notkins
authors.forename: R F
authors.initials: RF
authors.lastname: Rando
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D001483:Base Sequence; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000235:genetics*; D015497:HIV-1 / Q000276:immunology*; D004854:Herpesvirus 4, Human; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D008969:Molecular Sequence Data
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D015483:HIV Antibodies; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains