Background: Escherichia coli heat labile toxin B subunit (LTB) is one of the most popular oral vaccine adjuvants and intestine adsorption enhancers. It is often expressed as a fusion partner with target antigens to enhance their immunogenicity as well as gut absorbability. However, high expression levels of a fusion protein are critical to the outcome of immunization experiments and the success of subsequent vaccine development efforts. In order to improve the expression and functional assembly of LTB-fusion proteins using Saccharomyces cerevisiae, we compared their expression under culture conditions at a sub-physiological temperature 20 °C with their expression under a standard 30 °C.Results: The assembled expression of LTB-EDIII₂(LTB fused to the envelope domain III (EDIII) of Dengue virus serotype 2), which was expressed at the level of 20 µg/L in our previous study, was higher when the expression temperature was 20 °C as opposed to 30 °C. We also tested whether the expression and functional assembly of a difficult-to-express LTB fusion protein could be increased. The assembled expression of the difficult-to-express LTB-VP1 fusion protein (LTB fused to VP1 antigen of Foot-and-Mouth Disease Virus) dramatically increased, although the total amount of expressed protein was still lower than that of LTB-EDIII₂. Slight but significant increase in the expression of well-known reporter protein eGFP, which has previously been shown to be increased by cultivation at 20 °C, was also observed in our expression system. As no significant changes in corresponding transcripts levels and cell growth were observed between 20 °C and 30 °C, we infer that translation and post-translational assembly are responsible for these enhancements.Conclusions: The effects of lowering the expression temperature from 30 °C to 20 °C on protein expression and folding levels inS. cerevisiae, using several proteins as models, are reported. When heterologous proteins are expressed at 20 °C, a greater amount of (specially, more assembled) functional proteins accumulated than at 30 °C. Although further studies are required to understand the molecular mechanisms, our results suggest that lowering the expression temperature is a convenient strategy for improving the expression of relatively complexly structured and difficult-to-express proteins inS. cerevisiae.

Objectives: To explore Saccharomyces cerevisiae as an expression platform for dengue oral immune complex vaccine development. Results: Molecular engineering was applied to create a fusion gene construct (scEDIII-PIGS) consisting of a yeast codon optimized sequence encoding for a synthetic consensus dengue envelope domain III (scEDIII) followed by a modified IgG Fc domain (PIGS). Northern blot showed transcription of the target gene, with a temporal expression pattern similar to those from previous work. Western blot showed assembly of various immune complexes from monomer to hexamer. Partial purification of scEDIII-PIGS was also attempted to demonstrate the feasibility of yeast system for immune complex vaccine development. Approximately 1 mg of scEDIII-PIGS can be produced from 1 l culture. Conclusion: This work demonstrated for the first time that various immunocomplex structures of our target protein could be efficiently produced in S. cerevisiae for future application in developing oral and injectable vaccines against various pathogens.

In tropical and subtropical areas, peanuts are a very important legume crop that is widely cultivated for food and cooking oil. They are, however, extremely susceptible to a wide range of phytopathogens, particularly soil-borne fungi, which result in low yields and poor seed quality. This study aimed to express three Trichoderma asperellum SH16 42 kDa chitinase-encoding genes in peanut to improve their resistance to some soil-borne fungi. Chi42 is a synthetic, intronless, wild-type gene, whereas syncodChi42-1 and syncodChi42-2 are peanut codon-optimized synthetic genes. These genes were introduced into a local peanut strain through Agrobacterium-mediated transformation. Analysis of the transgenic peanut lines showed that chitinase-specific activities from the syncodChi42-1 and syncodChi42-2 genes were approximately 1.2 and 1.4 times higher than those of the wild-type gene, respectively. The engineered peanut plants also exhibited antifungal activity against the soil-borne pathogenic fungus Sclerotium rolfsii. The transgenic peanut lines transformed with the two synthetic genes have stronger antifungal activities than those transformed with the wild-type version, suggesting that they could be used as novel peanut lines to combat phytopathogenic fungi.

Nuclear insertions of mitochondrial DNA sequences (numts) can introduce errors in population genetic studies using mitochondrial DNA markers (mtDNA markers). Despite their prevalence in mammalian genomes and the potential risk of contamination with mtDNA markers, adequate precautions are often overlooked. When investigating the genetic diversity of Vietnam sheep, we found numt contamination in D-loop amplification. Consequentially, to continue, we conducted a comprehensive in silico survey of numts using the reference genomes from the NCBI database. We identified four so-called “mega-numts” that are not only highly similar in sequence to the mitogenome but also span a large part of it, thus posing a serious problem for amplifying authentic mtDNA markers. We demonstrated the existence of two numts in three randomly selected Vietnam sheep individuals. Based on the assumed mutation rate of the D-loop region, we proposed a sequence identity threshold to help distinguish authentic D-loop sequences from their corresponding numts when studying contemporary sheep populations. We hope this study can serve as a guideline for future research into Vietnam sheep using mtDNA markers.

Enhanced Green Fluorescent Protein (eGFP) shows much stronger fluorescence than its ancestor, Green Fluorescent Protein (GFP), thus has been widely applied as a reporter for biomedical research. In this study, we reported the expression of a synthetic codon optimized gene encoding eGFP in Escherichia coli (E. coli). The gene was cloned into two expression vectors, pQE30 and pColdII and the resulting recombinant vectors were transformed into E. coli M15 and BL21 De3 RIL codon plus strains, respectively. The expression levels of functional eGFP showed a temperature dependent pattern, in which lowering the induction temperature increased the amount of functional eGFP. Surprisingly, eGFP showed a phenomenon called auto-induction when E. coli TOP10 cells carrying recombinant pQE30 and pColdII were grown on Luria Broth plates. The recombinant eGFP showed robust stability even at room temperature, thus greatly facilitated its purification and handling. Mouse polyclonal antibodies were conveniently generated against the protein. Besides its potential application as a reporter gene in E. coli, the gene and its expression systems reported here are extremely useful as models for teaching recombinant DNA technology at undergraduate level.

The Phan Rang sheep, considered the sole indigenous breed of Vietnam, are primarily concentrated in the two central provinces of Ninh Thuan and Binh Thuan, with Ninh Thuan accounting for more than 90% of the country’s sheep population. These provinces are known for their high temperatures and frequent droughts. The long-standing presence of the Phan Rang sheep in these regions suggests their potential resilience to heat stress—a trait of increasing interest in the face of global climate change. Despite the breed’s significance, a critical knowledge gap hinders conservation and breeding programs. To address this, our study employed a two-pronged approach. First, we collected body conformational data to aid in breed identification. Second, we analyzed mitochondrial DNA (D-loop) and Y chromosome markers (SRY and SRYM18) to elucidate the maternal and paternal lineages. Among the 68 Phan Rang sheep analyzed for their D-loop, 19 belonged to mitochondrial haplogroup A, while 49 belonged to haplogroup B. The haplogroups can be subdivided into 16 unique haplotypes. All 19 rams surveyed for their paternal lineages belonged to haplotypes H5 and H6. These findings strongly support the hypothesis of dual origins for the Phan Rang sheep. This study presents the first genetic data for the Phan Rang breed, providing crucial insights for future research and conservation efforts.