Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphoma vaccination.
An expression system for rapid and standardized production of human recombinant immunoglobulin Fab fragments in E. coli was developed. Functional folding of the Fab fragments was accomplished by the dicistronic expression vector pFab.gammakappa containing specialized leader sequences to direct the immunoglobulin heavy and light chains to the periplasmic bacterial space. A C-terminal hexahistidine tag of the Fd chain facilitated metal affinity chromatography and purification to homogeneity as assessed by SDS PAGE and silver staining. Specific antigen recognition by a hybridoma-derived Fab fragment was indistinguishable from that of the corresponding monoclonal antibody. This protocol may be useful for analysis of the antigen specificity of human B cells and for convenient production of lymphoma-derived idiotype protein for vaccination strategies. To obtain unmodified immunoglobulin cDNA sequences from small human biopsies for insertion into pFab.gammakappa, oligo(dG)-tailed cDNA was amplified with an oligo(dC)- and nested mu or kappa constant region-specific primers. Using single sets of primers for each class of immunoglobulin transcripts, the products of this anchored PCR reflected the relative abundance of the starting cell population and permitted reliable identification of clonal, lymphoma-derived sequences for subsequent expression cloning.
Osterroth
F
F
Department of Hematology/Oncology, Freiburg University Medical Center, Hugstetter Strasse 55, D-79106, Freiburg, Germany.
Alkan
O
O
Mackensen
A
A
Lindemann
A
A
Fisch
P
P
Skerra
A
A
Veelken
H
H
D019496:Cancer Vaccines / Q000276:immunology*; D003001:Cloning, Molecular; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D016393:Lymphoma, B-Cell / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology; D014407:Tumor Cells, Cultured; D014611:Vaccination
D019496:Cancer Vaccines; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins