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Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major public health concern. Nucleocapsid (N) protein is the most abundant structural protein on SARS-CoV-2 virions and induces the production of antibodies at the early stage of infection. Large-scale preparation of N protein is essential for the development of immunoassays to detect antibodies to SARS-CoV-2 and the control of virus transmission. In this study, expression of water-soluble N protein was achieved through inducing protein expression at 25°C with 0.5 mM IPTG for 12 h. Western blot and ELISA showed that recombinant N protein could be recognized by sera collected from subjects immunized with Sinovac inactivated SARS-CoV-2 vaccine. Four monoclonal antibodies namely 2B1B1, 4D3A3, 5G1F8, and 7C6F5 were produced using hybridoma technology. Titers of all four monoclonal antibodies in ELISA reached more than 1.28×10 6.0. Moreover, all monoclonal antibodies could react specifically with N protein expressed by transfection of pcDNA3.1-N into BHK-21 cells in IPMA and IFA. These results indicated that water-soluble N protein retained high immunogenicity and possessed the same epitopes as that of native N protein on virions. In addition, the preparation of water-soluble N protein and its monoclonal antibodies laid the basis for the development of immunoassays for COVID-19 detection.

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BACKGROUND: Lanthipeptides are a rapidly expanding family of ribosomally synthesized and post-translationally modified natural compounds with diverse biological functions. Lanthipeptide structural and biosynthetic genes can readily be identified in genomic datasets, which provides a substantial repository for unique peptides with a wide range of potentially novel bioactivities. To realize this potential efficiently optimized heterologous production systems are required. However, only a few class I lanthipeptides have been successfully expressed using Escherichia coli as heterologous producer. This may be attributed to difficulties experienced in the co-expression of structural genes and multiple processing genes as well as complex optimization experiments. RESULTS: Here, an optimized modular plasmid system is presented for the complete biosynthesis for each of the class I lanthipeptides nisin and clausin, in E. coli. Genes encoding precursor lanthipeptides were fused to the gene encoding the mCherry red fluorescent protein and co-expressed along with the required synthetases from the respective operons. Antimicrobially active nisin and clausin were proteolytically liberated from the expressed mCherry fusions. The mCherry-NisA expression system combined with in vivo fluorescence monitoring was used to elucidate the effect of culture media composition, promoter arrangement, and culture conditions including choice of growth media and inducer agents on the heterologous expression of the class I lanthipeptides. To evaluate the promiscuity of the clausin biosynthetic enzymes, the optimized clausin expression system was used for the heterologous expression of epidermin. CONCLUSION: We succeeded in developing novel mCherry-fusion based plug and play heterologous expression systems to produce two different subgroups of class I lanthipeptides. Fully modified Pre-NisA, Pre-ClausA and Pre-EpiA fused to the mCherry fluorescence gene was purified from the Gram-negative host E. coli BL21 (DE3). Our study demonstrates the potential of using in vivo fluorescence as a platform to evaluate the expression of mCherry-fused lanthipeptides in E. coli. This allowed a substantial reduction in optimization time, since expression could be monitored in real-time, without the need for extensive and laborious purification steps or the use of in vitro activity assays. The optimized heterologous expression systems developed in this study may be employed in future studies for the scalable expression of novel NisA derivatives, or novel genome mined derivatives of ClausA and other class I lanthipeptides in E. coli.

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Hepatitis B core antigen (HBcAg) can self-assemble into virus-like particles (VLPs) when expressed in Escherichia coli. We optimized the different of the expression plasmid pBV220, including the ribosome bind site (RBS), spacer region, promoter and replication origin (ori), as well as the hbc gene dosage, to enhance HBcAg transcription and translation in E. coli. The optimized construct with a customized RBS6, 6 nt spacer, T7 promoter and pUCori significantly increased the levels of HBc36GFP fusion protein to 3.4-folds compared to the control. Thereafter, we substituted hbc36gfp gene with different copies of the hbc gene and tested the effects of gene dosage on HBcAg expression. The HBcAg-VLPs yield obtained using an engineered strain with three copies of hbc was 842.1 ± 46.8 µg/mL, which was 2.2-folds higher compared to that in the control strain. Thus, our study provides a simple and effective strategy for improving HBcAg expression in E. coli. Since the HBcAg-VLPs are promising carriers for presenting foreign antigen epitopes, an in vitro expression system that can generate high levels of HBcAg-VLPs can serve as a promising tool for developing novel HBV vaccines and drugs.

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The sweet-tasting protein brazzein offers considerable potential as a functional sweetener with antioxidant, anti-inflammatory, and anti-allergic properties. Here, we optimized a chemically defined medium to produce secretory recombinant brazzein in Kluyveromyces lactis, with applications in mass production. Compositions of defined media were investigated for two phases of fermentation: the first phase for cell growth, and the second for maximum brazzein secretory production. Secretory brazzein expressed in the optimized defined medium exhibited higher purity than in the complex medium; purification was by ultrafiltration using a molecular weight cutoff, yielding approximately 107 mg L-1. Moreover, the total media cost in this defined medium system was approximately 11% of that in the optimized complex medium to generate equal amounts of brazzein. Therefore, the K. lactis expression system is useful for mass-producing recombinant brazzein with high purity and yield at low production cost and indicates a promising potential for applications in the food industry.

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BACKGROUND: Hyaluronidase catalyzes the hydrolysis of hyaluronan polymers to N-acetyl-D-glucosamine and D-glucuronic acid. This enzyme is a dimer of identical subunits. Hyaluronidase has different pharmaceutical and medical applications. Previously, we produced a recombinant hyaluronidase antigenic fragment of Streptococcus pyogenes. OBJECTIVES: This study aimed to improve the protein production and purity of hyaluronidase recombinant protein from S. pyogenes. In addition, the enzymatic activity of this protein was investigated. MATERIALS AND METHODS: The expression of hyaluronidase antigenic fragments was optimized using IPTG concentration, time of induction, temperature, culture, and absorbance of 0.6-0.8-1 at 600 nm. Afterwards, the expressed proteins were purified and the enzymatic activity was assessed by turbid metric method. RESULTS: Data indicated that maximum protein is produced in OD = 0.8, 0.5 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG), 37ºC, NB 1.5x, without glucose, incubated for overnight. The enzymatic activity of the recombinant protein was similar to the commercial form of hyaluronidase. CONCLUSIONS: The results showed that an antigenic fragment of the recombinant hyaluronidase protein from S. pyogenes has a considerable enzymatic activity. It can be suggested to use it for medical purposes. In addition, applications of bioinformatics software would facilitate the production of a smaller protein with same antigenic properties and enzymatic activity.