Major Histocompatibility Complex Allele-Specific Peptide Libraries and Identification of T-Cell Mimotopes

Abstract

We descrrbe here a method for generating large libraries of random peptides that may be screened for T-cell antigens (1). These libraries make use of the discovery of sequence motifs common to the peptides bound to a particular major hlstocompatibility complex (MHC) molecule (2). The motifs consist of a restricted peptide length and two or three fixed amino acids required for peptide binding to MHC (anchor motifs) (2–4). Thus the libraries, comprised of a particular motif with the remaining amino acids randomized, are MHC allele-specific and are useful for T-cells restricted to MHC molecules with known anchor motifs. A degenerate oltgonucleotide encoding the random peptide is cloned into the prokaryotic expression vector pMAL-c, such that the peptides are expressed as C-terminal fusions to maltose binding protein (MBP) This allows for their purification by amylose column chromatography and factor X_a, restriction protease cleavage (5). Factor X_a, cleaves after the tetrameric recognition sequence Ile-Glu-Gly-Arg (IEGR), thus peptides are released without amino-terminal additions. Large preparations of complex pools of peptides may be fractionated by reverse-phase HPLC. By screening the individual fractions for T-cell reactivity, the fingerprint of a T-cell receptor’s (TCR) fine specificity can be visualized (1,6). The peptides can also be detected in factor X_a,-treated bacterial lysates when the antigen-expressing clone 1s represented at less than 10^*3. Thus, by performing successive rounds of screening, the clone of interest may be isolated and the sequence of the peptide (mimotope) determined by sequencing its cloned oligonucleotide (1).