We are developing a genetic approach to study with a single antibody the folding and topology of LamB, an integral outer membrane protein from Escherichia coli K-12 In addition, they suggest that region 236 is buried at the external face of the outer membrane and that region 219 is exposed to the periplasm. Including the 3 sites previously determined, 11 permissive sites are now available in LamB, including 3 at the cell surface and most probably at least 3 in the periplasm. We discuss the nature of such sites, the generalization of this approach to other proteins, and possible applications.Genetic fusions between proteins have proved to be useful tools in the study of a large number of biological problems. P-Galactosidase, alkaline phosphatase, and 1-lactamase have been used as reporter enzymes to study the location or the folding of proteins and for various biotechnological applications (for recent discussions, see references 7, 27, and 28). For topological studies, such fusions present at least two kinds of limitations. First, they usually delete the COOH-terminal end of the vehicle protein; this end can be critical for the normal biogenesis of the protein. Second, properties of the passenger enzyme (size, sequence, etc.) may alter the location or folding of the vehicle protein (see, for example, references 31 and 42). These two problems occur in the case of the outer membrane protein LamB (3, 4), which is why the topological information on this protein comes from other approaches (10, 21).LamB is representative of a class of integral membrane proteins, the porins (35), which are peculiar for at least two reasons. They are mostly hydrophilic, with no apparent hydrophobic transmembrane segment, and the structured regions are essentially composed of beta-strand segments. LamB plays a role in the penetration of maltose and maltodextrins through the outer membrane and is the cell surface receptor for a number of bacteriophages, including phage lambda; the active form is a trimer (reference 10 and references therein). In the absence of precise crystallo-* Corresponding author. graphic data, most of our knowledge of LamB organization relies on genetic, immunological, and biochemical data. On these bases, we have proposed a two-dimensional folding model that we would like to test and refine (9, 10).To circumvent the problems encountered with reporter enzymes, we started a new and complementary approach that consists of using as a reporter a small peptide corresponding to a foreign antigenic determinant (i.e., a reporter epitope) which we insert genetically into permissive sites of LamB (4,8; reviewed in reference 22). Such sites tolerate insertions without loss of most biological properties of the protein and therefore without major changes in its cellular location and structure. Permissive sites can be identified by a genetic method (8), and the hybrid proteins generated are then probed with a monoclonal antibody (MAb) against the reporter epitope. This approach led us to show that upon insertion after residues 153...
Three groups of four rhesus macaques were immunized twice, one month apart with purified recombinant HIV-1LAI gp160 in the presence of either alum, incomplete Freund's adjuvant (IFA), or SAF-1. Two months later, the animals were injected twice again with a synthetic peptide with the sequence of the principal neutralization determinant (PND) of the HIV-1LAI isolate mixed with the same adjuvants. All animals received a booster injection of gp160 and PND peptide at 6 months. This regimen of immunization induced in the SAF-1 and IFA groups a high-titer neutralizing antibody response that declined progressively over the course of the following 6 months. In contrast, only a weak response was observed in the alum group. Neutralizing antibody titers varied as anti-PND titers, suggesting that they were principally targeted to the PND. A shortened immunization protocol comprising two injections of gp160 at 0 and 1 month followed by one injection of PND peptide at 3 months is suggested as optimal for the induction of high titers of HIV-1 neutralizing antibodies in primates.
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