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An ovalbumin fusion strategy to increase recombinant protein secretion in chicken eggs.

ABSTRACT: Maternal secretion of recombinant proteins into chicken eggs may provide a viable approach for pharmaceutical production but remains limited by poor secretion efficiency through the membrane of oviduct cells, despite high expression levels. Here, we used site-specific integration of an EGFP fused to the OVAL gene by a rigid linker, (EAAAK)₃, at the endogenous ovalbumin locus in chicken primordial germ cells to generate OVAL-E3-EGFP transgenic chickens, with transgenic chickens expressing CMV immediate enhancer/β-actin-driven EGFP (CAG-EGFP) as a non-secreted control. In OVAL-E3-EGFP chickens, EGFP protein produced in maternal oviducts accumulates to high levels in eggs, but not in eggs of CAG-EGFP chickens. These results indicated that the secretion of foreign proteins can be substantially increased through fusion to the highly secreted endogenous ovalbumin. This study describes a basis for high yield recombinant protein expression in chicken eggs, enabling rapid and scalable production of numerous pharmaceutical proteins or metabolites.

SUBMITTER: Xie L

PROVIDER: S-EPMC10785457 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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An ovalbumin fusion strategy to increase recombinant protein secretion in chicken eggs.

Xie Long L Huang Zhenwen Z Lan Meiyu M Cao Yaqi Y Sun Lingling L Zhang Lang L Zuo Erwei E Lu Yangqing Y

Journal of biological engineering 20240111 1

Maternal secretion of recombinant proteins into chicken eggs may provide a viable approach for pharmaceutical production but remains limited by poor secretion efficiency through the membrane of oviduct cells, despite high expression levels. Here, we used site-specific integration of an EGFP fused to the OVAL gene by a rigid linker, (EAAAK)<sub>3</sub>, at the endogenous ovalbumin locus in chicken primordial germ cells to generate OVAL-E3-EGFP transgenic chickens, with transgenic chickens express ...[more]

PMID: 38212799

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Project description:Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

Project description:BackgroundUcp3 is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Ucp3 is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including PPARalpha, MyoD and the histone acetyltransferase p300. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII). This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, but also co-regulates a number of metabolic genes. In this study we in silico analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus and identified several putative response elements for Coup-TFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents.ResultsBy quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816-840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays.ConclusionTranscriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence for a function of Ucp3 in lipid metabolism, Coup-TFII may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism.

Project description:IntroductionExcretory/secretory products (ESPs) derived from helminths have been reported to effectively control allergic inflammation, which have better therapeutic prospects than live parasite infections. However, it remains unknown whether ESPs from schistosome eggs can protect against allergies, despite reports alleging that schistosome infection could alleviate disordered allergic inflammation.MethodIn the present study, we investigated the protective effects of ESPs from Schistosoma japonicum eggs (ESP-SJE) on asthmatic inflammation. Firstly, we successfully established an allergic airway inflammation model in mice by alum-adjuvanted ovalbumin (OVA) sensitization and challenge. ESP-SJE were administered intraperitoneally on days -1 and 13 (before sensitization), on day 20 (before challenge), and on days 21-24 (challenge phase).ResultsThe results showed that ESP-SJE treatment significantly reduced the infiltration of inflammatory cells, especially eosinophils into the lung tissue, inhibited the production of the total and OVA-specific IgE during OVA-sensitized and -challenged phases, respectively, and suppressed the secretion of Th2-type inflammatory cytokines (IL-4). Additionally, ESP-SJE treatment significantly upregulated the regulatory T cells (Tregs) in the lung tissue during OVA challenge. Furthermore, using liquid chromatography-mass spectrometry analysis and Treg induction experiments in vitro, we might identify nine potential therapeutic proteins against allergic inflammation in ESP-SJE. The targets of these candidate proteins included glutathione S-transferase, egg protein CP422 precursor, tubulin alpha-2/alpha-4 chain, actin-2, T-complex protein 1 subunit beta, histone H₄, whey acidic protein core region, and molecular chaperone HtpG.ConclusionTaken together, the results discussed herein demonstrated that ESP-SJE could significantly alleviate OVA-induced asthmatic inflammation in a murine model, which might be mediated by the upregulation of Treg in lung tissues that may be induced by the potential modulatory proteins. Therefore, potential proteins in ESP-SJE might be the best candidates to be tested for therapeutic application of asthma, thus pointing out to a possible new therapy for allergic airway inflammation.

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An ovalbumin fusion strategy to increase recombinant protein secretion in chicken eggs.

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An ovalbumin fusion strategy to increase recombinant protein secretion in chicken eggs.

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