Generating DNA sequences encoding tandem peptide repeats suitable for expression and immunological application

Abstract

Tandem repeats of single short peptide sequences are useful for many purposes. Here we describe a method called ligation-PCR to construct DNA sequences encoding numerous tandem peptide repeats that can stably produce such repeats in both prokaryotic and eukaryotic cells. The method employs double-strand target monomers consisting of a short peptide coding sequences. These sequences contain 3-bp cohesive overhangs to ensure correct repeat orientation and reading frame during ligation. The ligation products are PCR amplified and directly cloned into a new TA-cloning vector, pZeroT. Constructs containing tandem 10-amino-acid myc-tag peptide coding sequence repeats that ranged from approximately 0.45-1.2 kb, representing 15-40 copies of the corresponding peptide, were successfully obtained by this method. When one of the constructs was subcloned into prokaryotic vector pET-28 c (+) and eukaryotic vector rGHpcDNA3.0, and introduced into E. Coli and COS-7 cells, respectively, proteins containing tandem myc-tag peptide repeats were expressed with expected molecular weights. Purified proteins from E. Coli could successfully stimulate a peptide specific immune response. This method provides a means to manipulate peptides at the nucleic acid level, and can serve as the basis for biological peptide synthesis, epitope-specific antibody production, and epitope-based DNA vaccine construction.