In vivo and in vitro lipidation of recombinant immunogens for direct iscomincorporation

We have previously reported strategies for Escherichia coli production of r ecombinant immunogens fused to hydrophobic tags to improve their capacity t o be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (19 99) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immumogens as means to achieve i scom incorporation through hydrophobic interaction. For the in vivo lipidat ion strategy, a general expression vector was constructed encoding a compos ite tag consisting of a sequence (1pp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinit y chromatography. Upon expression in E. coli, fatty acids would be linked t o the produced gene products. To achieve in vitro lipidation, the target im munogen would be expressed in frame with an N-terminal His(6)-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment Delta SAG1 from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as mo del immunogen in this study. The two generated fusion proteins, 1pp-His(6)- ABP-Delta SAG1 and His(6)-ABP-Delta SAG1, both expressed at high levels (ap proximately 5 and 100 mg/l, respectively), could be recovered to high purit y by ABP-mediated affinity chromatography, and were evaluated in iscom-inco rporation experiments. The His(6)-ABP-Delta SAG1 fusion protein was associa ted to iscom matrix with pre-incorporated chelating lipid. Both fusion prot eins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/associati on. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-tite r antigen-specific antibody responses upon immunization of mice. For this p articular target immunogen, Delta SAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the pr eparation employing the in vitro lipidation strategy, indicating that Delta SAG1 was suboptimally folded