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 heat-to-tail arrangement provides a convenient way to obtain multimetric 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 BspMII which enables unidirectional insertion of the DNA fragments to be polymerized [Kim and Szylbalski, 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 repititive sequences from the Plasmodium falciparum malaria blood-stage antigen Pf155/RESA, was assemble 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-linke immunosorbent assay and immunofluorescence using the second fusion protein. © 1990.