Sugar Utilization-Associated Food-Grade Selection Markers in Lactic Acid Bacteria and Yeast.

This comprehensive review explores the development of food-grade selection markers in lactic acid bacteria and yeast; some of their strains are precisely defined as safe microorganisms and are crucial in the food industry. Lactic acid bacteria, known for their ability to ferment carbohydrates into lactic acid, provide essential nutrients and contribute to immune responses. With its strong fermentation capabilities and rich nutritional profile, yeast finds use in various food products. Genetic engineering in these microorganisms has grown rapidly, enabling the expression of enzymes and secondary products for food production. However, the focus is on ensuring safety, necessitating food-grade selection markers. Traditional antibiotic and heavy metal resistance selection markers pose environmental and health risks, prompting the search for safer alternatives. Complementary selection markers, such as sugar utilization markers, offer a promising solution. These markers use carbohydrates as carbon sources for growth and are associated with the natural metabolism of lactic acid bacteria and yeast. This review discusses the use of specific sugars, such as lactose, melibiose, sucrose, D-xylose, glucosamine, and N-acetylglucosamine, as selection markers, highlighting their advantages and limitations. In summary, this review underscores the importance of food-grade selection markers in genetic engineering and offers insights into their applications, benefits, and challenges, providing valuable information for researchers in the field of food microbiology and biotechnology.

A bispecific nanobody targeting the dimerization interface of epidermal growth factor receptor: Evidence for tumor suppressive actions in vitro and in vivo.

Targeting the dimer interface for the epidermal growth factor receptor (EGFR) that is highly conserved in the structure and directly involved in dimerization may solve the resistance problem that plagues anti-EGFR therapy. Heavy chain single domain antibodies have promising prospects as therapeutic antibodies. A bispecific nanobody was constructed based on previously screened humanized nanobodies that target the ß-loop at the EGFR dimer interface, an anti-FcγRIIIa (CD16) of natural killer cells (NK) nanobodies and anti-human serum albumin (HSA) nanobodies. The target gene was effectively expressed and secreted while controlled by promoter GAP in Pichia pastoris X33, and the expressed product was purified with a cation exchange and nickel chelation chromatography. The bispecific nanobody specifically bound to the surfaces of EGFR-overexpressed human epidermal carcinoma A431 cells and effectively inhibited tumor cell growth both in vitro and in vivo. In the A431 cell nude mouse xenograft model, the growth inhibition effect from the bispecific nanobody was significantly increased with the assistance of peripheral blood mononuclear cells (PBMCs), which was consistent with the results obtained in vitro, suggesting that there was an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. In addition, the intraperitoneal administration of bispecific nanobodies effectively reached tumor tissues in the shoulder dorsal region, but in significantly less distributed quantities than EGFR Dimer Nb77. To conclude, a bispecific nanobody targeting the EGFR dimer interface with ADCC effect was successfully constructed.

Nanobodies targeting the interaction interface of programmed death receptor 1 (PD-1)/PD-1 ligand 1 (PD-1/PD-L1).

Targeting the interaction interface is an effective strategy to obtain programmed death receptor 1 (PD-1)/PD-1 ligand 1 (PD-L1) nanobody blockers. To validate this strategy, the interaction interface between PD-1 and the PD-L1 extracellular domain were analyzed using Cn3D 4.1. The peptide PD-1125-136 located at the interface of PD-1 was selected as the antigen to screen nanobodies from a humanized nanobody phage display library. Six different nanobodies were screened, with molecular weights of 12 ∼ 13 kDa, excluding a single basic protein. The nanobody with the longest CDR3 region, termed PD-1-Nb-B20, was selected for further analysis. For mass production, the C-terminal His6-tagged nanobody coding sequence was optimized and cloned into pET-21b for over-expression under the T7 promoter in Escherichia coli BL21 (DE3). PD-1-Nb-B20 was expressed and pancreatic adenocarcinoma cells BxPC-3 over-expressing PD-L1 were selected for nanobody competitive inhibition assays. The purified nanobodies significantly inhibited PD-1 binding to the surface of target cells, indicating their ability to block the PD-1/PD-L1 interaction.

[Preparation and identification of single-chain fragment variable against epidermal growth factor receptor 3].

Objective To express, purify and identify the single-chain fragment variable (scFv) against human epidermal growth factor receptor 3 (HER3). Methods We searched NCBI for the light chain sequence and heavy chain sequence of anti-HER3 mAb LJM716 to construct the gene of scFv against HER3. The recombinant expression vector pGAPZαA-anit-HER3-scFv was constructed using the constitutive expression vector pGAPZαA and then electro-transformed into Pichia Pastoris X-33 to screen the strains with high expression of the protein of interest. After shaking flask fermentation, the supernatant was purified by hydrophobic chromatography and metal ion affinity chromatography. The purified product was identified by Western blotting and ELISA. Results The anti-HER3-scFv gene was successfully constructed and the strains with high expression of anti-HER3-scFv were obtained. The anti-HER3-scFv was purified to a purity of more than 95% by two-step chromatography, and the purified yield was 192 mg/L. Western blotting showed that the anti-HER3-scFv was correctly expressed and ELISA indicated that anti-HER3-scFv could specifically recognize HER3. Conclusion The anti-HER3-scFv has been successfully prepared.

Preparation and characterization of humanized nanobodies targeting the dimer interface of epidermal growth factor receptor (EGFR).

Epidermal growth factor receptor (EGFR) is an effective target for the treatment of many epithelial cancers. However, EGFR inhibitors have low clinical response rates and are prone to drug resistance arising from mutations and heterodimerization of EGFR. Therefore, targeting the highly conserved dimer interface of EGFR may be an effective strategy for improving the clinical response of anti-EGFR therapies. Nanobodies have significant advantages over conventional antibodies in terms of size, solubility, stability and cost-effectiveness. To investigate the feasibility of nanobodies targeting the dimer interface of EGFR as novel anticancer drugs, four nanobodies were screened from a commercial humanized nanobody phage antibody library using the EGFR237-267 peptide from the ß-hairpin loop of the dimer interface of EGFR as the antigen. A nanobody with an isoelectric point (pI) of 8.6, named EGFR dimer Nb77, was selected for further analysis of anticancer activities. EGFR dimer Nb77 was expressed in Escherichia coli Shuffle T7-B as a soluble (His)6-tagged protein and purified by a CM Sepharose column and a nickel-nitrilotriacetic acid (Ni-NTA) column. Purified EGFR dimer Nb77 could specifically bind to the surface of EGFR-overexpressing A431 cells in a dose-dependent and ligand-dependent manner, and this nanobody could effectively inhibit the growth of the tumour cells, with an inhibition rate similar to that of the monoclonal antibody EGFR dimer 5G9, which also targets the dimer interface of EGFR. This work is the first to prove that nanobodies targeting the dimer interface of EGFR have promising prospects as anticancer agents.

A monoclonal antibody targeting the dimer interface of epidermal growth factor receptor (EGFR).

Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of epithelial cancers. However, EGFR antagonists have low clinical response rates and frequently induce resistance mainly caused by the hypermutation of the extracellular and intracellular domains and the heterodimerization of EGFR. Dimerization plays a key role in the activation of the EGFR family of receptors. Thus, targeting the highly conserved dimer interface of EGFR maybe an attractive strategy for improving the clinical response of anti-EGFR therapies. In this work, we report a monoclonal antibody (mAb) called Antidimer 5G9 that targets the ß-hairpin within the dimer interface of EGFR, which is 100% homologous with mouse EGFR. Antidimer 5G9 belongs to the isotype IgG1 and bound with an affinity constant of 1.837×109L/mol. Just as designed and expected, Antidimer 5G9 could recognize and bind to human EGFRs or mouse EGFRs on the surface of human breast cancer cells of the MDA-MB-231 cell line or mouse Lewis lung cancer cells (LLC), effectively inhibiting growth and inducing apoptosis in the targeted cells. This work lays the foundation for further studies investigating the feasibility of this strategy for targeting the dimer interface of EGFR and developing novel anti-EGFR drugs.

B-cell epitope peptide vaccination targeting dimer interface of epidermal growth factor receptor (EGFR).

Epidermal growth factor receptor (EGFR) was the first receptor to be proposed as the target for cancer therapy, however, the anti-EGFR therapy showed a low response rate clinically. Dimerization plays a key role in the activation of EGFR, so targeting the conservative dimer interface probably improve the responses of anti-EGFR clinically. To evoke a high titers of antibodies (Abs) targeting the dimer interface of EGFR in patients, a chimeric peptide, comprising a linear B-cell epitope peptide from the highly conservative ß-hairpin loop of dimer interface of human EGFR (EGFR237-267) and a 'promiscuous' Th-cell epitope MVF from the measles virus fusion protein, was constructed. The construct was immunogenic and able to elicit high titers of peptide-specific Abs in both mice and rabbits, while both animals' EGFRs contain a sequence 100% homologous to EGFR237-267. The peptide-specific Abs could recognize and inhibit EGFR over-expressing epithelial cancer cells, such as epidermoid carcinoma A431 and Lewis lung cancer (LLC), furthermore, the binding of the Abs to the native EGFR on the surface of cells was ligand-dependent, which meant that these Abs did target the dimer interface of EGFR as designed, because the exposure of the interface needs ligand binding. The chimeric peptide immunization was able to significantly inhibit the growth of subcutaneously transplanted LLC cells in C57BL6 mice. Therefore, the MVF-EGFR237-267 construct represents a promising candidate for active anti-EGFR immunotherapy and provides a novel targeting strategy for the anti-EGFR therapy.

[Construction and immunogenicity analysis of tumor peptide vaccine P64k-EGFR(262-328); targeting the dimmerization interface of EGFR].

OBJECTIVE: To construct a tumor-specific peptide vaccine P64k-EGFR(262-328); targeting the dimerization interface of EGFR and analyze its immunogenicity in BALB/c mice. METHODS: The fusion gene of P64k-EGFR(262-328); was amplified by splicing overlap extension-PCR (SOE-PCR) and cloned into the pMD18-T vector. After double-enzyme cleavage and sequence analysis, the fusion gene was cloned into the expression vector pET-21b by digestion with Nde I and Hind III and then transformed into the BL21(DE3). After induced by IPTG to express, the fusion protein P64k-EGFR(262-328); was purified by source Q and Ni-NTA chromatography. BALB/c mice were immunized with the purified protein dissolved in double-distilled water emulsified (1:1) in Freuds' adjuvant, and their sera were tested for antibody titers by ELISA. RESULTS: The P64k-EGFR(262-328); gene of 2031 bp was acquired. SDS-PAGE showed that the recombinant protein was 70 kDa, expressed mainly in a soluble form. After two steps of column chromatography, the purity was over 95%. The peptide vaccine could elicit a high titer of more than 1:16 000 in BALB/c mice. CONCLUSION: The tumor-specific peptied vaccine P64k-EGFR(262-328); was constructed successfully, which could pay a good foundation for the further study on its function and application.

Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.

Separation of supercoiled from open circular forms of plasmid DNA, and biological activity detection.

To establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccination, a single anion-exchange chromatography (AEC) step was employed to purify supercoiled plasmid DNA (sc pDNA) from other isoforms and Escherichia coli impurities present in a clarified lysate. Two different size and conformation plasmids were used as model targets, and showed similar elution behavior in this chromatographic operation, in which sc pDNA was effectively separated from open circle plasmid DNA (oc pDNA) in a salt gradient. The process delivered high-purity pDNA of homogeneity of 95 ± 1.1% and almost undetectable levels of endotoxins, genomic DNA, RNA and protein, at a yield of 65 ± 8%. Furthermore, the transfection efficiency (29 ± 0.4%) was significantly higher than that (20 ± 0.1%) of a pDNA control. The present study confirms the possibility of using a single AEC step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.