Immunosilencing a Highly Immunogenic Protein Trimerization Domain

Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains, such as the widely used GCN4-based isoleucine zipper (IZ) and the T4 bacteriophage fibritin foldon (Fd) trimerization domains. We found that these domains induced potent anti-IZ or anti-Fd antibody responses in animals when fused to an HIV-1 envelope glycoprotein (Env) immunogen. To dampen IZ-induced responses, we constructed an IZ domain containing four N-linked glycans (IZN4) to shield the underlying protein surface. When fused to two different vaccine antigens, HIV-1 Env and influenza hemagglutinin (HA), IZN4 strongly reduced the antibody responses against the IZ, but did not affect the antibody titers against Env or HA. Silencing of immunogenic multimerization domains with glycans might be relevant for therapeutic proteins and protein vaccines.     

Protection against Helicobacter pylori infection by a trivalent fusion vaccine based on a fragment of urease B-UreB414

A multivalent fusion vaccine is a promising option for protection against Helicobacter pylori infection. In this study, UreB414 was identified as an antigenic fragment of urease B subunit (UreB) and it induced an antibody inhibiting urease activity. Immunization with UreB414 partially protected mice from H. pylori infection. Furthermore, a trivalent fusion vaccine was constructed by genetically linking heat shock protein A (HspA), H. pylori adhesin A (HpaA), and UreB414, resulting in recombinant HspA-HpaA-UreB414 (rHHU). Its protective effect against H. pylori infection was tested in BALB/c mice. Oral administration of rHHU significantly protected mice from H. pylori infection, which was associated with H. pylori-specific antibody production and Th1/Th2-type immune responses. The results show that a trivalent fusion vaccine efficiently combats H. pylori infection, and that an antigenic fragment of the protein can be used instead of the whole protein to construct a multivalent vaccine.     

Vaccine strategies in the treatment of low-grade non-Hodgkin lymphoma

Recent years have witnessed the development of a variety of promising immunotherapies for treating patients with B-cell non-Hodgkin's lymphomas. Each B lymphocyte expresses an immunoglobulin molecule that is the product of a unique combination of gene segments. B cell malignancy arises from one original B lymphocyte, and therefore all the members of a given lymphoma tumor population have the same unique immunoglobulin, which can serve as a target for immune therapy. When the idiotype (Id), or unique portion, of each immunoglobulin is used as a vaccine, antibodies and T cells can be induced and each can cause rejection of the tumor by the host. This special opportunity for tumor specificity is accompanied by the challenge of constructing a different vaccine for each patient. The first clinical trial of Id vaccination for lymphoma was initiated at Stanford University in 1988. Tumor cells obtained from lymph node sampling were fused with a myeloma cell line to generate a "hybridoma" producing large quantities of idiotype protein. Purified Id protein was then chemically coupled to keyhole limpet hemocyanin (KLH) and emulsified in an "oil-in-water" type immunologic adjuvant. The initial trial included patients with low-grade, follicular lymphoma, in first remission following chemotherapy. Among the first 32 vaccinated patients, roughly half (14/32) developed anti-Id immune responses. These were principally humoral responses rather than cellular responses. Long-term follow-up of these 32 patients has revealed that the development of an immune response is strongly correlated with prolonged freedom from disease progression interval and overall survival. Further trials have confirmed significant clinical benefit following Id vaccination. There is reason for excitement about the prospects for effective vaccine therapies for lymphoma as randomized Id vaccine trials commence and newer cell-based vaccine trials enter the clinic. As the clinical activity of lymphoma vaccines becomes established, it will be important to determine how to best integrate active vaccination approaches with standard therapeutic approaches.     

Soluble expression of Candida antarctica lipase B in Escherichia coli by fusion with Skp chaperone

Candida antarctica lipase B (CalB) is an industrially versatile enzyme, especially for biodiesel production and organic synthesis. Recombinant expression using the E. coli system has advantages, such as lower costs, easier handling, and higher number of clones that can be screened daily compared to expression using higher organism. But the expression of CalB in E. coli is not feasible because insoluble aggregates are formed and proteolytic degradation is known to occur during expression. In this study, fusion proteins were designed to express soluble CalB in E. coli. The periplasmic chaperone of E. coli, Skp was fused with CalB and this fusion protein showed a high solubility (yielding 82.5 ??g/mL). The fusion protein system can be applied to the rapid expression and evaluation of CalB variants for functional improvement.     

Expression,Polyubiquitination, and Therapeutic Potential of Recombinant E6E7 from HPV16 Antigens Fused to Ubiquitin

Ubiquitin–proteasome system plays an essential role in the immune response due to its involvement in the antigen generation and presentation to CD8⁺ T cells. Hereby, ubiquitin fused to antigens has been explored as an immunotherapeutic strategy that requires the activation of cytotoxic T lymphocytes. Here we propose to apply this ubiquitin fusion approach to a recombinant vaccine against human papillomavirus 16-infected cells. E6E7 multi-epitope antigen was fused genetically at its N- or C-terminal end to ubiquitin and expressed in Escherichia coli as inclusion bodies. The antigens were solubilized using urea and purified by nickel affinity chromatography in denatured condition. Fusion of ubiquitin to E6E7 resulted in marked polyubiquitination in vitro mainly when fused to the E6E7 N-terminal. When tested in a therapeutic scenario, the fusion of ubiquitin to E6E7 reinforced the anti-tumor protection and increased the E6/E7-specific cellular immune responses. Present results encourage the investigation of the adjuvant potential of the ubiquitin fusion to recombinant vaccines requiring CD8⁺ T cells.     

Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax

BACKGROUND: Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens. METHODS: In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63). RESULTS: Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22. CONCLUSION: VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.     

Heterodimeric Barnase-Barstar Vaccine Molecules: Influence of One versus Two Targeting Units Specific for Antigen Presenting Cells

It is known that targeting of antigen to antigen presenting cells (APC) increases immune responses. However, it is unclear if more than one APC-specific targeting unit in the antigenic molecule will increase responses. To address this issue, we have here made heterodimeric vaccine molecules that each express four different fusion subunits. The bacterial ribonuclease barnase and its inhibitor barstar interact with high affinity, and the barnase-barstar complex was therefore used as a dimerization unit. Barnase and barstar were fused N-terminally with single chain fragment variable (scFv)s targeting units specific for either MHC class II molecules on APC or the hapten 5-iodo-4-hydroxy-3-nitrophenylacetyl (NIP). C-terminal antigenic fusions were either the fluorescent protein mCherry or scFv³¹⁵ derived from myeloma protein M315. The heterodimeric vaccine molecules were formed both in vitro and in vivo. Moreover, the four different fused moieties appeared to fold correctly since they retained their specificity and function. DNA vaccination with MHC class II-targeted vaccine induced higher mCherry-specific IgG1 responses compared to non-targeted control. Since mCherry and MHC class II are in trans in this heterodimer, this suggests that heterodimeric proteins are formed in vivo without prior protein purification. Surprisingly, one targeting moiety was sufficient for the increased IgG1 response, and addition of a second targeting moiety did not increase responses. Similar results were found in in vitro T cell assays; vaccine molecules with one targeting unit were as potent as those with two. In combination with the easy cloning strategy, the heterodimeric barnase-barstar vaccine molecule could provide a flexible platform for development of novel DNA vaccines with increased potency.     

EGFR基因重组T7噬菌体疫苗抗Lewis肺癌的实验研究

本研究中制备了表达表皮生长因子受体(EGFR)部分肽段的基因重组T7噬菌体疫苗,并开展了诱导小鼠产生内源性抗EGFR抗体的实验性抗肿瘤作用研究。由T7噬菌体展示系统将7个经筛选的异种属(人源、鸡源)EGFR膜外区片段展示在其壳体次要头蛋白(P10B)上,用所制备的基因重组噬茵体疫苗免疫小鼠,免疫4W后皮下接种Lewis肺癌细胞,10d后分离瘤体并称重,观察各实验组的抗肿瘤效果。Western Blot检测重组的融合壳蛋白均有EGFR抗原性:高表达EGFR的A431 细胞与免疫3W的小鼠抗血清结合并被荧光二抗标记,流式细胞仪检测法确认有抗EGFR抗体产生;各实验组肿瘤均重统计结果显示,P-CL1-670组、P-cp1-130组、P-cp2-136组、P-cp3-145组、 P-cp4-142组与空白噬菌体组差异性显著。说明表达EGFR的基因重组噬菌体疫苗诱导产生的内源性抗体．在一定程度上抑制了EGFR阳性肿瘤的生长．为诱导型内源性抗EGFR抗体的肿瘤靶向治疗研究开辟了新的途径。     

EGFR基因重组T7噬菌体疫苗抗Lewis肺癌的实验研究

本研究中制备了表达表皮生长因子受体（EGFR）部分肽段的基因重组T7噬菌体疫苗,并开展了诱导小鼠产生内源性抗EGFR抗体的实验性抗肿瘤作用研究。由T7噬菌体展示系统将7个经筛选的异种属（人源、鸡源）EGFR膜外区片段展示在其壳体次要头蛋白（P10B）上,用所制备的基因重组噬菌体疫苗免疫小鼠,免疫4W后皮下接种Lewis肺癌细胞,10d后分离瘤体并称重,观察各实验组的抗肿瘤效果。WesternBlot检测重组的融合壳蛋白均有EGFR抗原性：高表达EGFR的A431细胞与免疫3W的小鼠抗血清结合并被荧光二抗标记．流式细胞仪检测法确认有抗EGFR抗体产生;各实验组肿瘤均重统计结果显示,P—CL1—670组、P—cp1-130组、P—cp2—136组、P—cp3—145组、P—cp4—142组与空白噬菌体组差异性显著。说明表达EGFR的基因重组噬菌体疫苗诱导产生的内源性抗体,在一定程度上抑制了EGFR阳性肿瘤的生长,为诱导型内源性抗EGFR抗体的肿瘤靶向治疗研究开辟了新的途径。     

Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli

Background

The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity; however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their applications. Immunization with an NP–M2e fusion protein expressed in Escherichia coli may represent an alternative strategy for the development of a universal influenza vaccine.

Methodology/Principal Findings

cDNA encoding M2e was fused to the 3′ end of NP cDNA from influenza virus A/Beijing/30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide gel almost completely protected the mice against a lethal (20 LD₅₀) challenge of heterologous influenza virus A/PR/8/34.

Conclusions/Significance

The NM2e fusion protein expressed in E. coli was highly immunogenic in mice. Immunization with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus. Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza vaccine.     

A new strategy for full-length Ebola virus glycoprotein expression in E.coli

Ebola virus(EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein(GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses.However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 ℃. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.     

Genetic fusion of chemokines to a self tumor antigen induces protective, T-cell dependent antitumor immunity

We converted a model, syngeneic, nonimmunogenic tumor antigen into a vaccine by fusing it with a proinflammatory chemokine. Two chemokines, interferon inducible protein 10 and monocyte chemotactic protein 3, were fused to lymphoma Ig variable regions (sFv). The sFv-chemokine fusion proteins elicited chemotactic responses in vitro and induced inflammatory responses in vivo. Furthermore, in two independent models, vaccination with DNA constructs encoding the corresponding fusions generated superior protection against a large tumor challenge (20 times the minimum lethal dose), as compared with the best available protein vaccines. Immunity was not elicited by controls, including fusions with irrelevant sFv; fusions with a truncated chemokine that lacked receptor binding and chemotactic activity; mixtures of free chemokine and sFv proteins; or naked DNA plasmid vaccines encoding unlinked sFv and chemokine. The requirement for linkage of conformationally intact sFv and functionally active chemokine strongly suggested that the mechanism underlying these effects was the novel targeting of antigen presenting cells (APC) for chemokine receptor-mediated uptake of antigen, rather than the simple recruitment of APC to tumor by the chemokine. Finally, in addition to superior potency, these fusions were distinguished from lymphoma Ig fusions with granulocyte-macrophage colony-stimulating factor or other cytokines by their induction of critical effector T cells.     

Delivering HIV Gagp24 to DCIR Induces Strong Antibody Responses In Vivo

Targeting dendritic cell-specific endocytic receptors using monoclonal antibodies fused to desired antigens is an approach widely used in vaccine development to enhance the poor immunogenicity of protein-based vaccines and to induce immune responses. Here, we engineered an anti-human DCIR recombinant antibody, which cross-reacts with the homologous cynomolgous macaque receptor and was fused via the heavy chain C-terminus to HIV Gagp24 protein (αDCIR.Gagp24). In vitro, αDCIR.Gagp24 expanded multifunctional antigen-specific memory CD4⁺ T cells recognizing multiple Gagp24 peptides from HIV-infected patient peripheral blood mononuclear cells. In non human primates, priming with αDCIR.Gagp24 without adjuvant elicited a strong anti-Gagp24 antibody response after the second immunization, while in the non-targeted HIV Gagp24 protein control groups the titers were weak. The presence of the double-stranded RNA poly(I:C) adjuvant significantly enhanced the anti-Gagp24 antibody response in all the groups and reduced the discrimination between the different vaccine groups. The avidity of the anti-Gagp24 antibody responses was similar with either αDCIR.Gagp24 or Gagp24 immunization, but increased from medium to high avidity in both groups when poly(I:C) was co-administered. This data provides a comparative analysis of DC-targeted and non-targeted proteins for their capacity to induce antigen-specific antibody responses in vivo. This study supports the further development of DCIR-based DC-targeting vaccines for protective durable antibody induction, especially in the absence of adjuvant.     

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.     

Electroporation as a "prime/boost" strategy for naked DNA vaccination against a tumor antigen

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetanus toxin, amplification of anti-tumor Ab, CD4(+), and CD8(+) T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8(+) T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8(+) T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.     

Dendritic cells: Potential role in cancer therapy

Dendritic cells (DC) are extremely potent antigen presenting cells, uniquely capable of sensitizing naive T cells to protein antigens and eliciting antigen specific immune responses. Studies of human DC isolated from peripheral blood indicate that these cells can be used to stimulate and expand antigen specific CD4+ and CD8+ T cells, in vitro. On the basis of these findings we have initiated pilot clinical studies to investigate the ability of DC pulsed ex vivo with tumor associated proteins to stimulate host anti-tumor immunity when re-infused as a vaccine. In the first such study DC pulsed with tumor derived idiotype protein were infused into patients with low grade malignant B cell lymphoma who had failed conventional chemotherapy. The majority of treated patients developed T cell mediated anti-idiotype immune responses and some of the patients experienced tumor regression. These results suggest that DC based immunotherapy is a potentially useful approach to B cell lymphoma and raises the possibility that the approach may prove useful in the treatment of other tumors as well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chloroplast expression of an HIV envelop-derived multiepitope protein: towards a multivalent plant-based vaccine

The development of a vaccine is still a priority in the fight against human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). Since conventional vaccine strategies have failed to provide a highly immunoprotective effect, approaches based on the rational design of vaccines composed of multiple HIV neutralizing epitopes have been proposed as potential vaccines. The aim of this study is to design a multiepitopic protein (Multi-HIV) carrying several neutralizing epitopes from both gp120 and gp41 as an effort to develop a new broad immunization scheme against HIV. This Multi-HIV was initially produced in a recombinant Escherichia coli strain either as a single protein or fused to glutathione-S-transferase. These proteins were purified by immobilized metal ion affinity chromatography and shown to be antigenic by positive reactivity in Western blot analyses using sera from HIV-positive patients for labeling. Since global immunization strategies are often limited by costs, platforms that require minimal processing are the priority in this field. Therefore, we explored the possibility of using transplastomic tobacco plants as an experimental model of a low cost plant-based vaccine against HIV. Transplastomic tobacco plants carrying the multi-HIV gene were developed and verified by PCR analyses. The expected Multi-HIV recombinant protein was localized in the chloroplast as proven first by confocal microscopy and subsequently by Western blot analysis. Tobacco-derived Multi-HIV protein was clearly able to evoke humoral responses in mice when orally administered without adjuvants. This report constitutes an effort to explore a new low-cost candidate that could have future implications on the development of affordable HIV vaccines.     

Heterogeneity of a murine B cell lymphoma. Isolation and characterization of idiotypic variants

mAb directed toward the idiotype of the 38C13 murine B cell lymphoma can be used to treat and cure a high percentage of mice challenged previously with an otherwise lethal dose of tumor cells. Tumors developing in animals despite antibody therapy were examined by immunofluorescence and found to demonstrate either loss of surface Ig, or expression of an altered idiotype that no longer bound the antibody used for treatment. Further immunofluorescence analysis of the variant tumors revealed individual patterns of cross-reactivity with anti-38C13 idiotype mAb other than that used for therapy. The variant tumor cells were fused to myeloma cells and hybrids were isolated which secreted large quantities of the altered idiotype proteins. Polyclonal antibodies and mAb prepared against the mutant proteins demonstrated cross-reactivity with the original 38C13 protein and its other variants. But the variants and wild type cells could be distinguished from each other by their patterns of reactivity with the panels of anti-idiotype antibodies. Differences in apparent m.w. were demonstrated in the L chains of each of the mutant proteins. Southern blot analysis of the H chain locus of these mutants established that they were all clonally related; however, the L chain loci were grossly different. Thus, rare cells with alteration in their Ig L chain genes and expressed proteins can give rise to idiotype variants in this B cell tumor.     

High-level production and purification of biologically active proteins from bacterial and mammalian cells using the tandem pGFLEX expression system

Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST π protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial production of biologically active recombinant proteins in E. coli and mammalian cells.     

One Hundred Seventy-Fold Increase in Excretion of an FV Fragment-Tumor Necrosis Factor Alpha Fusion Protein (sFV/TNF-α) from Escherichia coli Caused by the Synergistic Effects of Glycine and Triton X-100

To target tumor necrosis factor alpha (TNF-α) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene V_KLV_H–TNF-α, which encodes the secreted form of single-chain fusion protein sFV/TNF-α in Escherichia coli. sFV/TNF-α was secreted into the culture medium and purified by affinity chromatography. The production of the fusion protein in the culture medium under the optimal conditions of 30°C and 37 μmol of isopropyl-β-d-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard conditions of 37°C and 1 mmol of IPTG per liter. Fusion protein excretion into culture medium with 2% glycine, 1% Triton X-100, or both of these two chemicals was either 14-, 38-, or 170-fold higher, respectively than that without the two chemicals. The final yield of sFV/TNF-α was estimated to be 50 mg/liter. The loss of integrity of the cellular membrane may be a potential mechanism for enhancement of fusion protein production and excretion by treatment with glycine and Triton X-100. This study thus provides a practical, large-scale method for more efficient production of the heterologous fusion protein sFV/TNF-α in E. coli by using glycine and Triton X-100.The bacterium Escherichia coli has become a commonly used system for expression of heterologous recombinant proteins of interest in both biological research and the biotechnology industry. A variety of properties make the E. coli expression system attractive, namely, ease of genetic manipulation, efficient transformation, fast growth, simple fermentation, and favorable economics. Many recombinant proteins, including antibodies (15, 24) and single-chain fusion proteins (5, 25), have been successfully expressed in E. coli. Two major forms of heterologous proteins are usually expressed in this bacterium, i.e., insoluble and soluble. The former do not contain a signal peptide and are expressed in the cytoplasm and subsequently packaged into highly condensed inclusion bodies (2), while the latter have a signal peptide that is expressed in the cytoplasm and subsequently secreted into the periplasmic compartment. Although a high level of heterologous protein expression in inclusion bodies can be attained, these proteins are insoluble and therefore nonfunctional. A process of protein denaturing followed by a complex renaturing procedure must be conducted to obtain properly refolded functional proteins (3). However, the final yield of these soluble refolded proteins is usually very low, due mainly to protein aggregation resulting from the exposure of hydrophobic peptide regions (10).An alternative is to express the secretory form of heterologous proteins from the E. coli periplasmic fractions (23). Sometimes, however, the secreted heterologous proteins can leak from the periplasm into the culture medium, possibly due to the increased permeability of cellular membranes during long incubation periods (24). Secretion of these proteins into the E. coli periplasm is a useful ploy that can lead to the rapid isolation of recombinant proteins for biological evaluation. Its application on an industrial scale is limited by the general unavailability of efficient large-scale methods for the selective release of periplasmic proteins from the cell. Since it is easier to process the heterologous proteins in the culture medium than in the periplasmic fraction, various approaches have been developed to enhance the secretion of heterologous proteins of E. coli into culture media. These include (i) optimizing culturing conditions by modifying the temperature (4) or the concentration of isopropyl-β-d-thiogalactopyranoside (IPTG) (3), (ii) coexpression of molecular chaperones (13, 26), (iii) genetic modification of expression vectors (6, 11) or recombinant genes (8), and (iv) addition of chemicals such as glycine (9, 14).Recombinant single-chain fusion proteins have increasingly attracted attention in both research and clinical use due to their novel bifunctional activity and small size (5, 25). We have previously reported the construction and expression of single-chain fusion protein FV/TNF-α in inclusion bodies of E. coli (29). The fusion protein contains a single-chain FV fragment consisting of an immunoglobulin variable region of the heavy (V_H) (12.5 kDa) and light (V_K) (12.5 kDa) chains of the B72.3 antibody recognizing the human tumor-associated TAG72 antigen (28) and the tumor necrosis factor alpha (TNF-α) moiety (18 kDa). Previous studies have demonstrated that small antibody fragments such as FV (25 kDa) showed deeper, as well as more homologous, penetration of tumors by the molecule (19) and a higher localization index of tumors versus normal tissues (7) than the large intact antibody molecule (150 kDa). Therefore, this fusion protein (43 kDa) has the potential to efficiently target TNF-α to tumors expressing the TAG72 antigen for induction of active antitumor immune responses. Although the fusion protein retained its bifunctional activity after the process of denaturing and refolding, it still tended to aggregate especially in concentrations used in experimental animal model studies. This greatly limited its potential use as an antitumor therapeutic reagent. In this report, we describe the construction and expression of a secreted form of single-chain fusion protein sFV/TNF-α in E. coli. We also demonstrate dramatic enhancement of the excretion of this heterologous fusion protein from E. coli into culture media by the synergistic effect of glycine and Triton X-100.