Circular Economy and Sustainable Recovery of Taiwanese Tilapia (Oreochromis mossambicus) Byproduct-The Large-Scale Production of Umami-Rich Seasoning Material Application.

In this study, umami-rich seasoning powder was produced from the offcuts of Taiwanese tilapia (Oreochromis mossambicus) by cooking concentration and spray drying of granules while yielding an abundance of glutamic acid (0.23 mg/100 g), glycine (0.10 mg/100 g), aspartic acid (0.11 mg/100 g), lysine (0.10 mg/100 g), and 11 other aminic acids. It exhibited water content (3.81%), water activity (0.3), powder yields (68.83%), and a good water solubility index (99.89%), while the particle microstructure was a spherical powder. Additionally, it received the highest overall preference score (7.53) in the consumer-type sensory evaluation compared to commercially available seasonings. This study proves that offcuts may be part of the human diet after proper processing and can be widely used to flavor savory food. The producers involved could increase their economic returns while meeting the environmental challenges. The practical contribution could create incremental value for products to critical stakeholders at each point in the tilapia supply chain with an operational guide for transitioning from inefficient to innovative circular practices.

ERCC1 Overexpression Increases Radioresistance in Colorectal Cancer Cells.

Preoperative concurrent chemoradiotherapy (CCRT) is a standard treatment for locally advanced rectal cancer patients, but 20-30% do not benefit from the desired therapeutic effects. Previous reports indicate that high levels of ERCC1 reduce the effectiveness of cisplatin-based CCRT; however, it remains unclear as to whether ERCC1 overexpression increases radiation resistance. To clarify the correlation between ERCC1 levels and radiation (RT) resistance, we established two cell lines (HCT116-Tet-on and COLO205-Tet-on), induced them to overexpress ERCC1, detected cell survival following exposure to radiation, established HCT116-Tet-on and COLO205-Tet-on heterotopic cancer animal models, and detected tumor volume following exposure to radiation. We found that ERCC1 overexpression increased radiation resistance. After regulating ERCC1 levels and radiation exposure to verify the correlation, we noted that increased radiation resistance was dependent on ERCC1 upregulation in both cell lines. For further verification, we exposed HCT116-Tet-on and COLO205-Tet-on heterotopic cancer animal models to radiation and observed that ERCC1 overexpression increased colorectal cancer tumor radioresistance in both. Combined, our results suggest that ERCC1 overexpression may serve as a suitable CCRT prognostic marker for colorectal cancer patients.

HIF-1α Expression Increases Preoperative Concurrent Chemoradiotherapy Resistance in Hyperglycemic Rectal Cancer.

Purpose: Preoperative concurrent chemoradiotherapy (CCRT) is the standard treatment for locally advanced rectal cancer patients. However, the poor therapeutic efficacy of CCRT was found in rectal cancer patients with hyperglycemia. This study investigated how hyperglycemia affects radiochemotherapy resistance in rectal cancer. Methods and Materials: We analyzed the correlation between prognosis indexes with hypoxia-inducible factor-1 alpha (HIF-1α) in rectal cancer patients with preoperative CCRT. In vitro, we investigated the effect of different concentrated glucose of environments on the radiation tolerance of rectal cancers. Further, we analyzed the combined HIF-1α inhibitor with radiation therapy in hyperglycemic rectal cancers. Results: The prognosis indexes of euglycemic or hyperglycemic rectal cancer patients after receiving CCRT treatment were investigated. The hyperglycemic rectal cancer patients (n = 13, glycosylated hemoglobin, HbA1c > 6.5%) had poorer prognosis indexes. In addition, a positive correlation was observed between HIF-1α expression and HbA1c levels (p = 0.046). Therefore, it is very important to clarify the relationship between HIF-1α and poor response in patients with hyperglycemia receiving pre-operative CCRT. Under a high glucose environment, rectal cancer cells express higher levels of glucose transport 1 (GLUT1), O-GlcNAc transferase (OGT), and HIF-1α, suggesting that the high glucose environment might stimulate HIF-1α expression through the GLUT1-OGT-HIF-1α pathway promoting tolerance to Fluorouracil (5-FU) and radiation. In the hyperglycemic rectal cancer animal model, rectal cancer cells confirmed that radiation exposure reduces apoptosis by overexpressing HIF-1α. Combining HIF-1α inhibitors was able to reverse radioresistance in a high glucose environment. Lower HIF-1α levels increased DNA damage in tumors leading to apoptosis. Conclusions: The findings here show that hyperglycemia induces the expression of GLUT1, OGT, and HIF-1α to cause CCRT tolerance in rectal cancer and suggest that combining HIF-1α inhibitors could reverse radioresistance in a high glucose environment. HIF-1α inhibitors may be useful for development as CCRT sensitizers in patients with hyperglycemic rectal cancer.

Evaluation of Clove Extract for Drug Therapy of Ciliate Infection in Coral (Goniopora columna).

In recent years, ciliate infections have caused serious casualties to corals in the ocean. Infected corals die within a short period of time, which not only poses a threat to wild coral reefs, but also has a major impact on large scale aquaculture of coral. Clove is a kind of Chinese medicine with antifungal, antibacterial, antiviral, insecticidal, and other functions. Clove is a natural product. If it can be used in the treatment of coral ciliates, it will reduce this threat to the environment. The clove extract was diluted with sterile seawater to 500 ppm, 1500 ppm, 2500 ppm, 5000 ppm, 7500 ppm, and 10,000 ppm to carry out virulence test on ciliates. The results show that the LC50 value is 1500 ppm, which can cause the death of ciliates in 10 min without causing significant changes in G. columna SOD, CAT, chlorophyll a, and zooxanthellae. In addition, observation of tissue slices revealed that no ciliates and vacuum were found in the G. columna tissue after 10 min of medicated bathing. In summary, 1500 ppm of clove extract can be used for the treatment of coral ciliates.

Effects of LED Light Illumination on the Growth, Digestive Enzymes, and Photoacclimation of Goniopora columna in Captivity.

Goniopora columna is a stony coral valued for its reef-building potential and its unique appearance. Thus, identifying the optimal culture conditions for G. columna would enable efficient cultivation and prevent the illegal exploitation of marine resources. Light sources are crucial for the growth of corals because zooxanthellae provide them with basic nutrients through photosynthesis. Different corals and zooxanthellae have different photoacclimation characteristics; therefore, selecting a suitable light wavelength remains the key inhibitor of coral maintenance in marine aquariums. Accordingly, this study investigated the effects of different light wavelengths on G. columna. It was illuminated for 6 or 12 h a day under white light, yellow light, red light (LR), green light (LG), blue light (LB), or purple light (LP) for 8 weeks. During the experiment, R(R; i.e., a formula feed that combines sodium alginate, protein and probiotics) of 5% (w/v) of G. columna tissue and skeletal dry weight was fed every day. Coral polyps were counted, zooxanthellae density, chlorophyll a concentration, specific growth rates, and survival rates were calculated; polyp stretching and contractile behaviors were observed; and body composition and digestive enzyme activity were analyzed. LB or LP (but not LG or LR) illumination for at least 6 h per day significantly promoted the growth, survival, protein content, and protease activity of the G. columna specimens. Furthermore, coral polyp extension reached 100% after 30 min of LP and LB light irradiation. Although no significant differences in the zooxanthellae density or chlorophyll a concentration were noted under various light wavelengths, significant reductions were detected in the absence of light. To achieve energy-efficient coral aquaculture with regard to G. columna cultivation, 6 h of LB or LP illumination per day can improve the growth.

Separation and Identification of Resveratrol Butyrate Ester Complexes and Their Bioactivity in HepG2 Cell Models.

Resveratrol butyrate ester (RBE) complexes have demonstrated higher antioxidant capacity and anti-fat accumulation activity in previous studies. In this study, silica gel, high-performance liquid chromatography, and 1H nuclear magnetic resonance were used for separation and identification of RBE complex components. With the exception of resveratrol, five different structures of ester derivatives were separated from silica gel: 3,4'-di-O-butanoylresveratrol (ED2, 18.8%), 3-O-butanoylresveratrol (ED4, 35.7%), 4'-O-butanoylresveratrol (ED5, 4.4%), 3,5,4'-tri-O-butanoylresveratrol (ED6, 1.5%), and 3,5-di-O-butanoylresveratrol (ED7, 0.7%). Among the ester derivatives obtained, ED2 and ED4 were the main ester derivatives in the RBE complex. Thus, the cellular antioxidant activities of the RBE mixture, ED2, and ED4 were evaluated. Results showed that the antioxidant capacity of ED2 and ED4 was higher than that of the RBE mixture, demonstrating that the number and position of butyrate esterification sites are related to cell survival rate and antioxidant capacity. This study is the first to report the successful isolation, structural identification, and cellular biological antioxidant activity of RBE complex derivatives, which are key characteristics for the potential practical application of RBE complexes.

Effects of Ciliate Infection on the Activities of Two Antioxidant Enzymes (SOD and CAT) in Captive Coral (Goniopora columna) and Evaluation of Drug Therapy.

Ciliate infection is a serious parasitic disease of coral. Infected coral rots and dies in a short time. In addition to killing corals by infecting them in the oceans, ciliate infection also poses a threat to corals farmed on a large scale. In this study, two antioxidant enzymes (SOD and CAT) were used to judge the stress response in Goniopora columna after infection, and KCl and H2O2 were used to evaluate the therapeutic effect. The results showed that SOD and CAT increased during the early stage of infection but decreased with the extension of infection time. In terms of drug therapy, it was found that the treatment of ciliate infection with 1.5% of KCl had no significant effect on SOD and CAT of G. columna. The morphological changes of zooxanthellae, chlorophyll a, and coral were not significant. H2O2 leads to a stress response and polyp contraction. In conclusion, 1.5% of KCl can be used in the selection of drugs to treat ciliate infection.

Effect of cell-permeable grouper Manganese Superoxide Dismutase on environmental stress in fish.

Nitrite levels are generally high in high-density aquaculture. Nitrite is a potential stress-inducing factor and can cause oxidative stress because excessive reactive oxygen species (ROS) formation through nitrite induction cannot be scavenged by the endogenous antioxidant system, thus leading to cell damage or death. Manganese Superoxide Dismutase (MnSOD) is a highly efficient endogenous ROS scavenger that quenches mitochondrial ROS and protective against oxidative stress. To enhance the efficiency of MnSOD in removing ROS and reducing oxidative caused by nitrite, in this study, we cloned grouper MnSOD (gMnSOD) fused with a cell-penetrating peptide, TAT, to construct a TAT-gMnSOD fusion protein and assessed its potential to eliminate excess ROS induced by high nitrite concentrations and enhance the resistance of zebrafish to environmental stressors. Our results revealed that TAT-gMnSOD penetrated the grouper fin (GF-1) cells, scavenged nitrite-induced intracellular ROS, and enhanced cell viability on NaNO2 treatment. Furthermore, pretreatment of zebrafish with TAT-gMnSOD fusion protein reduced the MDA content and increased the survival rate. In addition, the TAT-gMnSOD fusion protein reduced 2-phenoxyethanol toxicity and attenuated excessive anesthesia among zebrafish. In conlusion, our cell-permeable TAT-gMnSOD fusion protein effectively counters oxidative stress, prevents environmental stress-induced damage, and increases aquaculture benefits.

Heterologous expression of bacterial CotA-laccase, characterization and its application for biodegradation of malachite green.

Malachite green (MG) is used as fungicide/parasiticide in aquaculture, its persistence is detrimental as it exhibits carcinogenic effects to aquatic organisms. Bacterial laccase evaluated as the best enzyme at extreme condition for aquatic MG removal. Study aims to increase laccase concentration, CotA-laccase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Optimal catalysis for purified CotA-laccase were at pH 5.0, 60 °C, and 1 mM of (2,2-azino-di-[3-ethylbenzothiazoline-sulphonate-(6)]) with Km and Kcat 0.087 mM and 37.64 S-1 respectively. MG biodegradation by CotA-laccase in clam and tilapia pond wastewaters and cytotoxic effect of biodegraded products in grouper fin-1 cells were determined. MG degradation by CotA-laccase was equally efficient, exhibiting upto 90-94% decolorization at freshwater and saline conditions and treated solution was non-toxic to GF-1 cells. Thus, recombinant-CotA-laccase could be an environmentally-friendly enzyme for aquaculture to remove MG, thereby effective to reduce its accumulation in aquatic organisms and ensuring safe aquaculture products.

Developing a Virus-Binding Bacterium Expressing Mx Protein on the Bacterial Surface to Prevent Grouper Nervous Necrosis Virus Infection.

Grouper nervous necrosis virus (GNNV) infection causes mass grouper mortality, leading to substantial economic loss in Taiwan. Traditional methods of controlling GNNV infections involve the challenge of controlling disinfectant doses; low doses are ineffective, whereas high doses may cause environmental damage. Identifying potential methods to safely control GNNV infection to prevent viral outbreaks is essential. We engineered a virus-binding bacterium expressing a myxovirus resistance (Mx) protein on its surface for GNNV removal from phosphate-buffered saline (PBS), thus increasing the survival of grouper fin (GF-1) cells. We fused the grouper Mx protein (which recognizes and binds to the coat protein of GNNV) to the C-terminus of outer membrane lipoprotein A (lpp-Mx) and to the N-terminus of a bacterial autotransporter adhesin (Mx-AIDA); these constructs were expressed on the surfaces of Escherichia coli BL21 (BL21/lpp-Mx and BL21/Mx-AIDA). We examined bacterial surface expression capacity and GNNV binding activity through enzyme-linked immunosorbent assay; we also evaluated the GNNV removal efficacy of the bacteria and viral cytotoxicity after bacterial adsorption treatment. Although both constructs were successfully expressed, only BL21/lpp-Mx exhibited GNNV binding activity; BL21/lpp-Mx cells removed GNNV and protected GF-1 cells from GNNV infection more efficiently. Moreover, salinity affected the GNNV removal efficacy of BL21/lpp-Mx. Thus, our GNNV-binding bacterium is an efficient microparticle for removing GNNV from 10‰ brackish water and for preventing GNNV infection in groupers.

Enhancement of tumor tropism of mPEGylated nanoparticles by anti-mPEG bispecific antibody for ovarian cancer therapy.

Ovarian cancer is highly metastatic, with a high frequency of relapse, and is the most fatal gynecologic malignancy in women worldwide. It is important to elevate the drug susceptibility and cytotoxicity of ovarian cancer cells, thereby eliminating resident cancer cells for more effective therapeutic efficacy. Here, we developed a bispecific antibody (BsAb; mPEG × HER2) that can easily provide HER2+ tumor tropism to mPEGylated liposomal doxorubicin (PLD) and further increase the drug accumulation in cancer cells via receptor-mediated endocytosis, and improve the cytotoxicity and therapeutic efficacy of HER2+ ovarian tumors. The mPEG × HER2 can simultaneously bind to mPEG molecules on the surface of PLD and HER2 antigen on the surface of ovarian cancer cells. Simply mixing the mPEG × HER2 with PLD was able to confer HER2 specificity of PLD to HER2+ ovarian cancer cells and efficiently trigger endocytosis and enhance cytotoxicity by 5.4-fold as compared to non-targeted PLD. mPEG × HER2-modified PLD was able to significantly increase the targeting and accumulation of HER2+ ovarian tumor by 220% as compared with non-targeted PLD. It could also significantly improve the anti-tumor activity of PLD (P < 0.05) with minimal obvious toxicity in a tumor-bearing mouse model. We believe that the mPEG × HER2 can significantly improve the therapeutic efficacy, potentially reduce the relapse freqency and thereby achieve good prognosis in ovarian cancer patients.

Use of syngeneic cells expressing membrane-bound GM-CSF as an adjuvant to induce antibodies against native multi-pass transmembrane protein.

Membrane antigens (mAgs) are important targets for the development of antibody (Ab) drugs. However, native mAgs are not easily prepared, causing difficulties in acquiring functional Abs. In this study, we present a platform in which human mAgs were expressed in native form on cell adjuvants made with membrane-bound cytokines that were then used immunize syngeneic mice directly. The membrane-bound cytokines were used as immune stimulators to enhance specific Ab responses against the desired mAgs. Then, mAgs-expressing xenogeneic cells were used for Ab characterization to reduce non-specific binding. We established cell adjuvants by expressing membrane-bound cytokines (mIL-2, mIL-18, or mGM-CSF) on BALB/3T3 cells, which were effective in stimulating splenocyte proliferation in vitro. We then transiently expressed ecotropic viral integration site 2B (EVI2B) on the adjuvants and used them to directly immunize BALB/c mice. We found that 3T3/mGM-CSF cells stimulated higher specific anti-EVI2B Ab response in the immunized mice than the other cell adjuvants. A G-protein coupled receptor (GPCR), CXCR2, was then transiently expressed on 3T3/mGM-CSF cell adjuvant to immunize mice. The immune serum exhibited relatively higher binding to xenogeneic 293 A/CXCR2 cells than 293 A cells (~3.5-fold). Several hybridoma clones also exhibited selective binding to 293 A/CXCR2 cells. Therefore, the cell adjuvant could preserve the native conformation of mAgs and exhibit anti-mAg Ab stimulatory ability, providing a more convenient and effective method to generate functional Abs, thus possibly accelerating Ab drug development.

Pharmacological inhibition of bacterial ß-glucuronidase prevents irinotecan-induced diarrhea without impairing its antitumor efficacy in vivo.

Irinotecan (CPT-11), a first-line chemotherapy for advanced colorectal cancer, causes serious diarrhea in patients receiving treatment. The underlying mechanism has been shown that the active metabolite of CPT-11, SN-38, is metabolized to the inactive metabolite SN-38 glucuronide (SN-38 G) during hepatic glucuronidation, and subsequently is exported into the intestine, where SN-38 G is hydrolyzed by bacterial ß-glucuronidase (ßG) to be SN-38, thus leading to intestinal toxicity. Thus, inhibition of the intestinal bacterial ßG activity is expected to prevent CPT-11-induced diarrhea. However, the effects of such inhibition on serum pharmacokinetics of SN-38, the key determinant of CPT-11 treatment, are uncertain. Here, we determined the effects of a potent E. coli ßG (eßG)-specific inhibitor pyrazolo[4,3-c]quinoline derivative (TCH-3562) for the potential use in preventing CPT-11-induced diarrhea. TCH-3562 exhibited efficacious inhibitory potency of endogenous ßG activity in two anaerobes, Eubacteriumsp. and Peptostreptococcus anaerobius. Oral administration of TCH-3562 also effectively reduced the bacterial ßG activity in mice intestine. Moreover, pharmacokinetic analysis of TCH-3562 revealed a relatively low amount of TCH-3562 was detected in the plasma whereas the majority of TCH-3562 was found in the feces. Importantly, co-treatment of CPT-11 and TCH-3562 did not decrease active SN-38 level in mice plasma. Finally, we established that TCH-3562 as an adjuvant treatment showed protective effects on CPT-11-induced diarrhea and had no negative effects on the therapeutic efficacy of CPT-11 in tumor-bearing mice. Therefore, inhibition of the intestinal bacterial ßG activity by the specific inhibitor, TCH-3562, is promising to prevent CPT-11-induced diarrhea while maintaining its anti-tumor efficacy that may have clinical potentials for the treatment with CPT-11.

Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of poly-protein G-expressing cell-based microplates.

The sensitivity of traditional enzyme-linked immunosorbent assays (ELISAs) is limited by the low binding avidity and heterogeneous orientation of capture antibodies coated on polystyrene-based microplates. Here, we developed a highly sensitive ELISA strategy by fixing poly-protein G-expressing cells on microplates to improve the coating amount and displayed orientation of capture antibodies. One or eight repeated fragment crystallisable (Fc) binding domains of protein G are stably expressed on the surface of BALB/c 3T3 cells (termed 1pG cells or 8pG cells), which then act as highly antibody-trapping microparticles. The 8pG cells showed higher antibody-trapping ability than the 1pG cells did. The antibody-coating amount of the 8pG cell-based microplates was 1.5-23 times and 1.2-6.8 times higher than that of traditional polystyrene-based and commercial protein G-based microplates, respectively. The 8pG cell-based microplates were then applied to an anti-IFN-α sandwich ELISA and an anti-CTLA4 competitive ELISA, respectively, and dramatically enhanced their detection sensitivity. Importantly, direct coating unpurified capture antibody produced by mammalian cells did not impair the antigen-capturing function of 8pG cell-based microplates. The 8pG cell-based microplates exhibited a significant improvement in antibody-coating amount and preserved the homogeneous orientation of capture antibodies, making them a potential replacement for traditional microplates in various formats of ELISAs.

Author Correction: Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays.

The sensitivities of solid-phase immunoassays are limited by the quantity of detection antibodies bound to their antigens on the solid phase. Here, we developed a poly-protein G-expressing bacterium as an antibody-trapping microparticle to enhance the signals of immunoassays by increasing the accumulation of detection antibodies on the given antigen. Eight tandemly repeated fragment crystallisable (Fc) binding domains of protein G were stably expressed on the surface of Escherichia coli BL21 cells (termed BL21/8G). BL21/8G cells showed a higher avidity for trapping antibodies on their surface than monomeric protein G-expressing BL21 (BL21/1G) cells did. In the sandwich enzyme-linked immunosorbent assay (ELISA), simply mixing the detection antibody with BL21/8G provided a detection limit of 6 pg/mL for human interferon-α (IFN-α) and a limit of 30 pg/mL for polyethylene glycol (PEG)-conjugated IFN-α (Pegasys), which are better than that of the traditional ELISA (30 pg/mL for IFN-α and 100 pg/mL for Pegasys). Moreover, the sensitivity of the Western blot for low-abundance Pegasys (0.4 ng/well) was increased by 25 folds upon mixing of an anti-PEG antibody with BL21/8G cells. By simply being mixed with a detection antibody, the poly-protein G-expressing bacteria can provide a new method to sensitively detect low-abundance target molecules in solid-phase immunoassays.

Development of Membrane-Bound GM-CSF and IL-18 as an Effective Tumor Vaccine.

The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (P<0.05) and enhanced tumor protective efficacy (P<0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies.

Impediments to enhancement of CPT-11 anticancer activity by E. coli directed beta-glucuronidase therapy.

CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/ßG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/ßG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/ßG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/ßG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity.

Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.

Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.