A general strategy for polymerization, assembly and expression of epitope-carrying peptides applied to the Plasmodium falciparum antigen Pf155/RESA.
Polymerization of DNA fragments in a head-to-tail arrangement provides a convenient way to obtain multimeric expression of a specific gene product, e.g., epitope-carrying peptides for immunological studies. A novel technique for the polymerization and assembly of peptides has been developed, involving the use of the class-IIS restriction enzyme BspMI which enables unidirectional insertion of the DNA fragments to be polymerized [Kim and Szybalski, Gene 71 (1988) 1-8]. One or several DNA fragments are polymerized in subsequent steps, using in vitro DNA polymerization, and the obtained gene constructs containing several repeats are screened and sequenced using polymerase chain reaction techniques. Using a two-step polymerization strategy a peptide, comprising two repetitive sequences from the Plasmodium falciparum malaria blood-stage antigen Pf155/RESA, was assembled and subsequently synthesized in Escherichia coli. Two different fusion proteins suitable for affinity purification were produced using a dual affinity system. Rabbits were immunized with one of the fusion proteins and the antibody response was analyzed by the enzyme-linked immunosorbent assay and immunofluorescence using the second fusion protein.
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
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Ståhl
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Sjölander
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M
Hansson
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Nygren
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Uhlén
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics* / Q000276:immunology; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping
D000953:Antigens, Protozoan; D003062:Codon; D000939:Epitopes; D046911:Macromolecular Substances; D004247:DNA