Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli.

Abstract

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 ...

Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+CD8+Dendritic Cells for Induced Long-Term Protective Immunity

“…After blocking, serial dilutions of serum or saliva samples were added in duplicate. The starting dilution of serum was 1:2 5 , while that of saliva was 1:2 1 . The plates were incubated for 4 h at room temperature, washed, and then incubated with horseradish peroxidase-conjugated goat anti-mouse heavy chain ␥-, ␥1-, ␥2a-, ␥2b-, ␥3-, or ␣-specific antibodies (Southern Biotechnology Associates, Birmingham, AL) at 4°C for 20 h. Finally, 2,2Ј-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) with H 2 O 2 (Moss, Inc., Pasadena, MD) was added for color development.…”

“…MBP is used as a fusion partner for recombinant protein expression to improve the yield and to facilitate the purification of fusion proteins (5,47). Furthermore, the stability and solubility of a passenger protein can be improved by fusing it to MBP (12).…”

Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker

“…Thrombin, however, cleaves the amino acid sequence LVPR/GS between R and G, which leaves two amino acids (GS), called an overhang, at the amino terminus of the target protein. All three enzymes have been shown to cleave proteins at non-canonical sites [6][7][8][9][10], which negates the advantages afforded by the carrier protein.To circumvent these problems, two additional proteases, rhinovirus 3C protease (called PreScission protease) and the nuclear inclusion protease encoded by tobacco etch virus (TEV), have been introduced to the marketplace. While these proteases may exhibit a higher specificity than the previous enzymes, both enzymes leave overhangs on the target protein.…”

“…Thrombin, however, cleaves the amino acid sequence LVPR/GS between R and G, which leaves two amino acids (GS), called an overhang, at the amino terminus of the target protein. All three enzymes have been shown to cleave proteins at non-canonical sites [6][7][8][9][10], which negates the advantages afforded by the carrier protein.…”

Expression and Purification of Glutathione Transferase-Small Ubiquitin-Related Modifier-Metallothionein Fusion Protein and Its Neuronal and Hepatic Protection against d-Galactose-Induced Oxidative Damage in Mouse Model

“…To date, there remains no study using E. coli to produce large amounts of soluble MT, mainly because of the small molecular mass of MTs (Butt et al, 1989;Zhou et al, 2000;Hong et al, 2001). In addition, MTs have toxic effects on the host strains and inhibit their growth because of the existence of more than 20 sulfhydryl groups in its amino acid composition (Butt et al, 1989;Zhou et al, 2000;Hong et al, 2001).…”