Despite the availability of efficient transcription and translation signals, some heterologous gene products are not adequately expressed when introduced into prokaryotes and eukaryotes. An expression system has been established in Escherichia coli to increase the yield of cloned gene products, where the C terminus of ubiquitin was fused to the N terminus of unstable or poorly expressed proteins. Fusion of ubiquitin to yeast metallothionein or to the a subunit of the adenylate cyclase-stimulatory GTP-binding protein increased the yield from undetectable to 20% of the total cellular protein. A ubiquitin-Na-protein hydrolase has been partially purified from rabbit reticulocytes; this enzyme faithfully cleaves the junction peptide bond between the C-terminal Gly-76 of ubiquitin and the fusion protein. The increased yield of cloned gene products is very likely due to increased stability and/or more efficient translation of the fusion proteins. Possible mechanisms for the augmentation of ubiquitin fusion-protein expression in prokaryotes and eukaryotes are discussed. protein is cleaved with factor Xa to release the pure protein (7). The disadvantages of this system are that desired N termini cannot be constructed and often the protein is cleaved at multiple sites by factor Xa.Ubiquitin, a 76-amino acid polypeptide, is the most conserved protein known among eukaryotes (8). Ubiquitin and the ubiquitin pathway are not present in prokaryotes. In eukaryotes, ubiquitin exists as a free molecule or with its C terminus covalently linked to the E-amino groups of lysine in other proteins (8). In addition, natural ubiquitin fusionprotein genes have been cloned and sequenced from eukaryotes in which the N-terminal end of the protein is ubiquitin followed by a C-terminal extension of 52-80 amino acids (9-11). Ubiquitin-p-galactosidase (12) and ubiquitin-metallothionein (UB-MT) (13) fusion proteins and human ubiquitin C-terminal extension proteins (HUBCEPs) (10) have been expressed in yeast, and the ubiquitin moiety was rapidly processed except when the first amino acid of the extension protein was proline.We have observed that the fusion of genes to ubiquitin sequences greatly increased their yield (up to 20% oftotal cell protein) in E. coli. Expression of UB-MT and UB-Gsa [where Gsa is the a subunit of the GTP-binding stimulatory protein of adenylate cyclase (14)] were studied in E. coli. The unfused proteins were extremely unstable or not translated efficiently, whereas fusion with ubiquitin stabilized the proteins (and perhaps enhanced their translation), thus increasing their yield. We have also characterized and partially purified an enzyme, ubiquitin-Na-protein hydrolase (UaPH), from rabbit reticulocytes that faithfully cleaves these ubiquitin fusion proteins to release ubiquitin and the authentic protein. The utility of this system to increase the yield of unstable or poorly expressed proteins in E. coli is discussed.
MATERIALS AND METHODSConstruction of Ubiquitin Fusion-Protein Expression Vectors. Ubiquitin genes ...
The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella‐containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane‐bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP‐bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63‐containing late endosomes. Nischarin is recruited to the SCV in a Rab14‐dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners—Rac1, Rab14 and Rab9 GTPases—reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.
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