Asparaginase of Escherichia coli, a tetramer of identical subunits, was tested as a vector to display linear peptides on the surface of each enzyme subunit. A recombinant gene encoding a chimeric protein composed of asparaginase, a tetanus toxin peptide (TTP) spacer (831-854 fragment), and the foreign cholesteryl ester transfer protein C-terminal fragment (CETPC) was expressed and targeted to the periplasm of E. coli. The purified chimeric enzyme exhibited approximately 83% activity of the native enzyme, allowing the rapid screening of recombinant clones. In contrast, an asparaginase-CETPC fusion protein without the TTP spacer produced only about 23% activity of the native enzyme. Rats immunized with bacterial cells containing the chimeric enzymes induced CETP-specific immunoresponse. In contrast, rats inoculated with the cells expressing asparaginase only did not generate specific anti-CETP antibodies. Our study showed that asparaginase of E. coli was an effective carrier for displaying foreign peptides or epitopes. Moreover, the use of the TTP spacer appeared to play a critical role in maintaining the catalytic activity of the chimeric enzymes by redirecting the foreign CETPC peptide to the surface of the enzyme. The chimeric enzyme constructs fusing asparaginase with foreign peptides via a TTP spacer could be utilized as a rapid pepscan technique for antigen epitope mapping. The fusion protein of asparaginase-TTP-CETPC could also be useful for the development of a vaccine against atherosclerosis.