General expression vectors for production of hydrophobically tagged immunogens for direct iscom incorporation.

A new general strategy for the production of recombinant protein immunogens has been investigated. The rationale involves the production of a recombinant immunogen as fused to a composite tag comprising one domain suitable for affinity purification and a hydrophobic tag designed for direct incorporation through hydrophobic interaction of the affinity-purified immunogen into an adjuvant system, in this case immunostimulating complexes (iscoms). Three different hydrophobic tags were evaluated: (i) a tag denoted IW containing stretches of hydrophobic isoleucine (I) and tryptophan (W) residues; (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus; and (iii) a tag denoted PD designed to be pH-dependent in such a way that an amphiphatic alpha-helix would be formed at low pH. As an affinity tag, an IgG-binding domain Z derived from Staphylococcus aureus protein A (SpA) was used, and a malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as a model immunogen in this study. Three different fusion proteins, IW-Z-M5, MI-Z-M5 and PD-Z-M5, were produced in Escherichia coli, and after affinity purification these were evaluated in iscom-incorporation experiments. Two of the fusion proteins, IW-Z-M5 and MI-Z-M5 were found in the iscom fraction following preparative ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. Furthermore, these iscom preparations were demonstrated to induce efficient M5-specific antibody responses upon immunization of mice, confirming successful incorporation into iscoms. The implications of these results for the design and production of subunit vaccines are discussed.

Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding.

The cholera toxin B (CTB) subunit has been used extensively in vaccine research as a carrier for peptide immunogens due to its immunopotentiating properties, where coupling has been obtained either by genetic fusion or chemical conjugation. For genetically fused immunogens both N- and C-terminal fusions have been used. Only shorter extensions have previously been evaluated and in some reports these fusions have impaired the biological functions of CTB, such as the ability to form pentamers and to adhere to its cell receptor, the GM1 ganglioside. Here we report the first systematic study where the same fusion partner has been used for either C-terminal, N-terminal or dual fusions to CTB. The serum albumin binding region (BB, approximately 25 kDa) from streptococcal protein G, which is known to fold independently of N- or C-terminal fusions, was selected as fusion partner. The three fusion proteins CTB-BB, BB-CTB and BB-CTB-BB were expressed in Escherichia coli, where they were efficiently secreted to the periplasmic space, and could be purified by affinity chromatography on human serum albumin (HSA) columns. The CTB fusion proteins were compared for their ability to form pentamers, by gel electrophoresis and size-exclusion chromatography, and it was concluded that all three fusion proteins were able to pentamerize. Interestingly, the C-terminal fusion to CTB showed most efficient pentamerization, while the dual fusion was much less efficient. Purified pentamer fractions from all three fusions where found to react to a monoclonal antibody described to react only to pentameric forms of CTB. In addition, the purified pentamer fractions were analyzed in an enzyme-linked immunosorbent assay (ELISA) for their ability to bind GM1, and it was found that the C-terminal fusion (CTB-BB) showed significant GM1-binding, but that also the N-terminal and dual CTB fusion proteins bound GM1, although less efficiently. The implications of the results for the design and use of CTB fusion proteins as subunit vaccines are discussed.

Upstream strategies to minimize proteolytic degradation upon recombinant production in Escherichia coli.

Proteolytic degradation of recombinant proteins represents a major problem related to production of gene products in heterologous hosts. Recombinant DNA technology offers several alternative strategies for stabilization of expressed gene products. These strategies can often give dramatic stabilization effects and can be combined with strategies involving optimization of fermentation conditions or downstream processing schemes. In this review, various genetic approaches to improve the stability of recombinant proteins will be discussed, including (i) choice of host cell strain, (ii) product localization, (iii) use of gene fusion partners, and (iv) product engineering. In addition, the solubility of the gene product can be influenced by factors such as growth temperature, promoter strength, fusion partners, and site-directed changes. Altogether, a battery of approaches can be used to obtain stable gene products.

Hydrophobicity engineering to increase solubility and stability of a recombinant protein from respiratory syncytial virus.

Site-directed mutagenesis has been employed to engineer the hydrophobic properties of a 101-amino-acid fragment from the human respiratory syncytial virus (RSV) major glycoprotein (G protein). When this protein was produced in Escherichia coli, more than 70% of the gene product was found as inclusion bodies, and the product recovered from the soluble fraction was severely degraded. Substitution of two cysteine residues for serine residues, did not significantly change the solubility or stability of the gene product. In contrast, a dramatic increase in both solubility and stability was achieved by multiple engineering of hydrophobic phenylalanine residues. As compared to the non-engineered protein, the fraction of soluble protein in vivo could be increased from 27% to 75%. Surprisingly, this effect was accompanied by a remarkable increase in stability. The in vitro solubility of the purified gene products was similarly increased approximately fivefold. Structural studies using circular dichroism suggest that the two engineered fragments have a distribution of secondary-structure elements similar to the non-engineered fragment. In addition, the two engineered G-protein variants were demonstrated to be at least in part antigenically authentic to the non-engineered gene product. These results demonstrate that engineering of hydrophobic residues can be used as a tool to increase the solubility and proteolytic stability of poorly soluble and labile proteins.

Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy.

A dual affinity fusion approach has been used to study the expression and secretion of labile recombinant proteins in Escherichia coli. Here we show that three small eukaryotic proteins (human proinsulin, a thioredoxin homologous domain of rat protein disulfide isomerase, and the extracellular domain of the alpha 1.2-chain of a human T-cell receptor) are stabilized in vivo using a dual affinity fusion strategy, where the gene encoding the desired product is fused between two genes encoding two different affinity domains. Relatively high yields of full-length product were obtained for all three proteins as compared to when fused to a single fusion partner. Despite the use of a signal peptide, significant amounts of the disulfide protein isomerase and T-cell receptor gene products were maintained in the cytoplasm, while the proinsulin fusion was efficiently secreted to the periplasm. Interestingly, the E. coli heat shock proteins DnaK and GroEL were associated with the fusion proteins isolated from the cytoplasm.

Differential degradation of a recombinant albumin-binding receptor in Escherichia coli.

The degradation in Escherichia coli of the recombinant serum-albumin-binding receptor derived from streptococcal protein G was investigated using a dual-affinity fusion approach. The proteolytic degradation of the receptor was characterized when fused to human proinsulin and human secretin. Several cleavages occurred at sequences not normally regarded as proteolytically sensitive, such as the dipeptide sequences Ile-Gly, Val-Ser and Ser-Ala. Depending on the fusion partner, large differences in the degradation of the albumin-binding domain were observed. Thus, susceptibility to proteolysis of a recombinant protein can be affected by a neighbouring domain.

Solid phase in vitro mutagenesis using plasmid DNA template.

Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions. More than 80% mutants were obtained in both a single and a double primer approach. No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coli host cell. The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a site-specific restriction followed by a fill-in reaction. This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols.

Differential stability of recombinant human insulin-like growth factor II in Escherichia coli and Staphylococcus aureus.

Recombinant human insulin-like growth factor II (IGF-II), produced as a soluble extracellular fusion protein, was shown to be proteolytically degraded in Escherichia coli. In contrast, the fusion protein secreted from Staphylococcus aureus was stable and the full length product could be recovered by affinity chromatography. After site specific cleavage of the fusion protein, soluble IGF-II with biological activity was obtained without refolding procedures. These results demonstrate that a eukaryotic protein unstable in E. coli can be stabilized by expression in a Gram positive host. The full-length fusion protein from S. aureus was used to characterize the protease responsible for the degradation in E. coli. Biochemical and genetic analysis suggests a specific degradation by the outer membrane protease (OmpT).