We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 10⁸-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutatedin vitroby acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.

An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli . The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the F _v fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The F _v fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant F _v fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the F _v fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the F _v fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2D^b, chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8⁺ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2D^b in association with peptide GP33–41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8⁺ T cells were GP33 tetramer⁺ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8⁺ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.