Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphomavaccination

An expression system for rapid and standardized production of human recombi nant immunoglobulin Fab fragments in E. coli was developed. Functional fold ing of the Fab fragments was accomplished by the dicistronic expression vec tor pFab.gamma kappa 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 ch romatography and purification to homogeneity as assessed by SDS PAGE and si lver staining. Specific antigen recognition by a hybridoma-derived Fab frag ment was indistinguishable from that of the corresponding monoclonal antibo dy. This protocol may be useful for analysis of the antigen specificity of human B cells and for convenient production of lymphoma-derived idiotype pr otein for vaccination strategies. To obtain unmodified immunoglobulin cDNA sequences from small human biopsies for insertion into pFab.gamma kappa, ol igo(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 cl ass of immunoglobulin transcripts, the products of this anchored PCR reflec ted the relative abundance of the starting cell population and permitted re liable identification of clonal, lymphoma-derived sequences for subsequent expression cloning. (C) 1999 Elsevier Science B.V. All rights reserved.