Generating DNA sequences encoding tandem peptide repeats suitable for expression and immunological application.
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.
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Cell Biology, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, People's Republic of China.
Hongwei
H
Hou
Zhiqian
Z
Zhang
Wei
W
Zhao
Jinyan
J
Hou
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D010455:Peptides / Q000235:genetics* / Q000276:immunology*; D016133:Polymerase Chain Reaction / Q000379:methods; D020449:Repetitive Sequences, Amino Acid; D020080:Tandem Repeat Sequences
D010455:Peptides