Epitope-specific antibody and suppression of autoantibody responses against a hybrid self protein.

This study addresses the relationship of epitope-specific Ab responses and alternative autoantibody responses in a model system in which an antigenized self protein serves as the carrier for a defined heterologous B cell epitope. Ubiquitin, a nonimmunogenic self protein, was engineered to present heterologous B and T cell epitopes in the recombinant molecule. Fusion to the C terminus introduced a universal T cell epitope from a Mycobacterium tuberculosis Ag. The B cell epitope was created by inserting a 12-residue loop sequence of HIV-1 gp120 at a surface-exposed position of ubiquitin. These modifications preserved the ubiquitin fold, allowing a new conformational epitope to be presented among native self epitopes. Mice immunized with the hybrid protein bearing only the mycobacterial T cell epitope elicited a strong autoantibody response to native ubiquitin. In contrast, antisera elicited against hybrid ubiquitin presenting the HIV B cell epitope reacted specifically with the foreign epitope but not with native ubiquitin. Absence of autoantibody in the response was attributed to poor competition of autoreactive B cells for limiting T cell help. Both types of responses were associated with Th responses to defined epitopes of the ubiquitin hybrid protein. These results may have implications for a tolerance mechanism dependent on B-T cell cooperation.

High-level expression and efficient recovery of ubiquitin fusion proteins from Escherichia coli.

The transition in growth and induction of bacterial cultures expressing recombinant proteins from the laboratory bench to the pilot scale for production has been performed successfully for several ubiquitin fusion-expressing clones. Increased protein turnover and decreased metabolic efficiency of Escherichia coli at high cell densities are often responsible for failures in fermenter cultures. Current data indicate that (1) yields in shaker flask cultures are directly scalable to a 10 L fermenter, (2) higher cell densities actually augment the specific yield of ubiquitin fusion proteins, (3) an in vivo heat shock during fermentation increases the ubiquitin fusion yield, which provides an initial separation step in the fermenter, and (4) the ubiquitin fusion expression clone makes at least 3-fold more total protein, including host proteins, than the parent strain of E. coli. A series of three fermentations was performed, using the model strain, with varied temperature shift protocols. These fermentations showed that a maximal heat shock of 12 degrees C (from 30 to 42 degrees C), initiated simultaneously with induction, gave a maximal specific yield (over 90% of the total soluble protein by densitometry, 709 mg/L by protein assay), in which the recoverable ubiquitin fusion product comprised 16% of the wet weight of the cell paste. These results illustrate the enormous potential of ubiquitin fusion technology for the economical production of peptides, even in a 10 L fermenter.

Identification and characterization of the ribosomal RNA-encoding genes in Clavibacter xyli subsp. cynodontis.

Clavibacter xyli subsp. cynodontis (Cxc) is a xylem-inhabiting bacterial endophyte of Bermudagrass. This organism is classified with Gram-positive, high G + C content, coryneform-actinomycete bacteria. Southern-blot analysis showed that Cxc contains only one copy of the ribosomal RNA-encoding genes (rRNA). A clone containing the rRNA genes was isolated from a genomic library of Cxc DNA cloned in the lambda EMBL3 vector. The gene cluster was partially sequenced, revealing the gene order 5'-16S-23S-5S-3', similar to that found in other prokaryotes. Low-resolution S1 mapping suggested multiple transcription start points of the rRNA operon.