Cell Protein Production Research Articles

Rhodotorula paludigena CM33 cultivation process development for high β-carotene single cell protein production

Rhodotorula yeast has potential in terms of biotechnology for the production of various compounds for the environmental, industrial, and medical sectors. It is well known to generate carotenoid pigments that are beneficially distinguished. This investigation aimed to optimize the growth of Rhodotorula paludigena CM33 and its production of β-carotene. Response Surface Methodology was employed to optimize temperature, ammonium sulfate, and ferric chloride concentrations in a 5L-bioreactor batch culture. The optimal conditions of 30 °C, 6.2 g/L of ammonium sulfate, and 75.0 mg/L of ferric chloride provided biomass of 17.71 g/L, a specific growth rate of 0.22 h−1, and β-carotene concentration of 40.29 mg/L. Upon scaling up the cultivation process, the 22L-bioreactor displayed similar growth patterns to those of the 5L-bioreactor. However, the 22L-bioreactor exhibited higher product yields in a shorter time period. The maximum biomass production of 18.96 ± 1.31 g/L and the maximum β-carotene production of 42.81 ± 0.61 mg/L were achieved at 28 h. This scaling-up strategy resulted in improved production efficiency. Additionally, the nutritional value of the cells was evaluated, suggesting their potential as animal feed supplements. R. paludigena CM33 demonstrated a β-carotene single-cell protein content, making it a valuable source for aquaculture and livestock feed. These findings highlight the potential of this yeast strain for large-scale production of β-carotene and single-cell protein, offering nutritional benefits for animal feed.

Conversion of Orange and Pineapple Fruit Peel Waste into Single Cell Protein Using Saccharomyces Cerevisiae

The biological treatment of fruit wastes into single-cell protein has the potential to address the global protein shortage problem by producing a cost-effective product for food and feed. Pollution may be reduced if the substrate was made from the leftovers from the food processing industry. Herein, the submerged fermentation method was to make single-cell protein from pineapple and orange peels. Results indicated that fruit wastes analysed were highly hydrolysable. Proximate analysis of fruit wastes protein content of orange peel (10.73±0.02%) was significantly higher (P<0.05) than the protein content of pineapple peels (7.50±0.016%). When Saccharomyces cerevisiae was cultivated on fruit hydrolysate medium (FHM) without supplementation, the proportion of protein in single-cell protein was substantially lower, which produced (41.50±0.05% and 37.97±0.006%) total carbohydrate with (29.73±0.031% and 34.50±0.4%) crude protein, in both pineapple and orange medium respectively. The introduction of glucose to the supplemented fruit hydrolysate medium (GSFHM), increased the level of protein (45.50±0.031 and 37.73±0.011%) within the yeast cell, the comparison of treatment showed P<0.05, thus we can conclude that there is significant difference in the overall comparison of treatments in most of the parameters. The ITS2 region sequence analysis of Saccharomyces cerevisiae MA851180, revealed 99% identity with Saccharomyces cerevisiae. Thus, Saccharomyces cerevisiae produces higher amount of Single Cell Protein by submerged fermentation of pineapple peel hydrolysate as compared to orange peel hydrolysate. Hence, the single cell protein production by yeast depends on the growth substrates or media composition. SCP from yeast using fruit waste can serve as cheaper alternative to the conventional protein gotten from plant and animal and by selecting the most suitable prospective microbe and cost-effective technique.

Single Cell Protein Production by Isolated And Identified Bacteria Using Crude Oil As A Carbon Source

Single Cell Protein Production by Isolated And Identified Bacteria Using Crude Oil As A Carbon Source

The effects of AtEXO70E2 overexpression on the secretion and production of recombinant proteins in tobacco BY-2 suspension cells

The effects of AtEXO70E2 overexpression on the secretion and production of recombinant proteins in tobacco BY-2 suspension cells

M6A-modified miR-143-3p inhibits epithelial mesenchymal transition in bronchial epithelial cells and extracellular matrix production in lung fibroblasts by targeting Smad3

BackgroundAirway epithelial cells epithelial mesenchymal transition (EMT) and lung fibroblasts extracellular matrix (ECM) production are the key steps in airway remodeling. Our previous study demonstrated that miR-143-3p has the ability to impede airway smooth muscle cell proliferation and ECM deposition. However, the function of miR-143-3p in airway epithelial cells and lung fibroblasts remains unclear. MethodsCell viability was determined using MTT method, while cell migration was evaluated through scratch assay. EMT and ECM proteins were detected by western blot, RT-qPCR, and ELISA. To determine the level of miR-143-3p m6A methylation, we employed the meRIP-qPCR assay. Additionally, the binding of miR-143-3p with Smad3 were projected by bioinformatics and validated by dual luciferase reporter assays. ResultsIt was discovered that the expression of miR-143-3p were lower in both asthma patients and TGF-β1-treated human bronchial epithelial 16HBE cells and human lung fibroblast HPF cells. Upregulation of miR-143-3p restrained 16HBE cell migration, and decreased EMT mesenchymal markers and increased epithelial markers. And upregulation of miR-143-3p impaired cell viability and ECM protein production in HPF cells. Mechanistically, interfering with METTL3 resulted in decreased m6A modification of miR-143-3p and led to lower levels of miR-143-3p. Moreover, miR-143-3p were verified to directly target and downregulate Smad3. Upregulation of Smad3 attenuated the effects of miR-143-3p on cell EMT and ECM production. ConclusionMiR-143-3p inhibits airway epithelial cell EMT as well as lung fibroblast ECM production by downregulating Smad3. Therefore, miR-143-3p may be a promising target to reduce airway remodeling in asthma.

New insights into self-structure induction in poly (rA) by Quinacrine through non-classical intercalation: Spectroscopic and theoretical perspectives

Self-structure induction in a single stranded polyriboadenylic acid [poly (rA)] is an auspicious physiological phenomenon which switches off protein production in tumor cells. In the present study, the self-structure induction process in poly (rA) moiety was thoroughly investigated using various steady state and time resolved techniques. Optical melting pattern directly evidenced the formation of self-structured assembly in single stranded poly (rA) upon complexation with quinacrine. Further, UV-absorption spectroscopic studies revealed that quinacrine binds to poly (rA) in co-operative fashion and the indication of intercalative mode of binding first came out with the involvement of around two base pairs of poly (rA) in the complexation. Experimental observations established the unconventional or non-classical intercalation of quinacrine molecule inside self-structured duplex poly (rA) moiety. This complexation was accompanied with negative enthalpy change and positive entropy change; suggesting strong van der Waals and the H-bonding interactions as the major governing forces in the complexation. Moreover, ionic strength dependent binding study established that the non-polyelectrolytic forces were the dominating forces. Further, the photo physical behavior of QN was authenticated using time dependent density functional theory (TDDFT) where both the ground and excited states were exploited.

Single cell protein and oil production from solid cocoa fatty acid distillates co-fed ethanol

The use of solid lipid sidestreams have been overlooked as a feedstock for the production of microbial biomass for food and feed applications and little to no recent work has examined the utilization of solid fatty acid distillates (FADs), which are a significant residue from vegetable oil processing. Yarrowia lipolytica and Rhodosporidium toruloides cultivated on cocoa fatty acid distillates (CFAD) generated final cell dry weight values > 40 g/L, with strong productivity (3.3 g/L·h) and rich protein (>45%) and lipid content (>25%). Interestingly, microbial oils were > 65% unsaturated fatty acids, compared < 20% unsaturated content in FAD. Importantly, to overcome mass-transfer limitations associated with bioconversion of solid lipid residues, ethanol was applied as a co-substrate to solubilize FAD residues. Here, FAD residues from cocoa deodorization have been demonstrated to be high energy feedstocks that represent an attractive substrate for the production of both single cell protein and oil (SCPO).

Cost-Effective Protein Production in CHO Cells Following Polyethylenimine-Mediated Gene Delivery Showcased by the Production and Crystallization of Antibody Fabs.

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes.

Integrating spheroid-on-a-chip with tubeless rocker platform: A high-throughput biological screening platform.

Spheroid-on-a-chip platforms are emerging as a promising in-vitro model that enables screening of the efficacy of biologically active ingredients. Generally, the supply of liquids to spheroids occurs in the steady flow mode with the use of syringe pumps; however, the utilization of tubing and connections, especially for multiplexing and high-throughput screening applications, makes spheroid-on-a-chip platforms labour- and cost-intensive. Gravity-induced flow using rocker platforms overcomes these challenges. Here, we develop a robust gravity-driven technique to culture arrays of cancer cell spheroids and dermal fibroblast spheroids in a high-throughput manner using a rocker platform. We benchmark the efficiency of the developed rocker-based platform to syringe pumps for generating multicellular spheroids and their use for screening biologically active ingredients. We study cell viability, internal spheroid structure, as well as the effect of vitamin C on the production of collagen type I and fibronectin by dermal fibroblast spheroids. We demonstrate that the rocker-based platform not only offers comparable or enhanced performance in terms of cell viability, spheroids formation, and protein production by dermal fibroblast spheroids but also, from a practical perspective it offers a smaller footprint, requires a lower cost, and offers an easier method for handling. These results support the application of rocker-based microfluidic spheroid-on-a-chip platforms for in-vitro screening in a high-throughput manner with industrial scaling-up opportunities. This article is protected by copyright. All rights reserved.

Spatial translatomics pinpoints protein production in cells

Spatial translatomics pinpoints protein production in cells

Enhanced bypass of PD-L1 translation reduces the therapeutic response to mTOR kinase inhibitors

Increased PD-L1 expression in cancer cells is known to enhance immunosuppression, but the mechanism underlying PD-L1 upregulation is incompletely characterized. We show that PD-L1 expression is upregulated through internal ribosomal entry site (IRES)-mediated translation upon mTORC1 inhibition. We identify an IRES element in the PD-L1 5'-UTR that permits cap-independent translation and promotes continuous production of PD-L1 protein despite effective inhibition of mTORC1. eIF4A is found to be a key PD-L1 IRES-binding protein that enhances PD-L1 IRES activity and protein production in tumor cells treated with mTOR kinase inhibitors (mTORkis). Notably, treatment with mTORkis invivo elevates PD-L1 levels and reduces the number of tumor-infiltrating lymphocytes in immunogenic tumors, but anti-PD-L1 immunotherapy restores antitumor immunity and enhances the therapeutic efficacy of mTORkis. These findings report a molecular mechanism for regulating PD-L1 expression through bypassing mTORC1-mediated cap-dependent translation and provide a rationale for targeting PD-L1 immune checkpoint to improve mTOR-targeted therapy.

Weak promoters to drive selection marker expression: Improvement of cell line development process for therapeutic protein production in CHO-K1 cells.

Chinese Hamster Ovary cells have been widely used as host cells for production of recombinant therapeutic molecules. Cell line development is a decisive step, which must be carried out with an efficient process. In particular, degree of selection stringency is an important parameter for identification of rare, high-producing cell lines. In the CHOZN® CHO K1 platform, selection of top-producing clones is based on puromycin resistance, whose expression is driven by Simian Virus 40 Early (SV40E) promoter. In this study, novel promoters have been identified to drive expression of selection marker. Decrease of transcriptional activity compared to SV40E promoter was confirmed by RT-qPCR. Selection stringency was increased, as seen by decreased surviving rate of transfected mini-pools and longer recovery duration of transfected bulk pools. Several promoters led to a 1.5-fold increase of maximum titer and a 1.3-fold increase of mean specific productivity of the monoclonal antibody over the clone generation. Expression level was maintained stable over long term cultivation. Finally, productivity increase was confirmed on several monoclonal antibodies and fusion proteins. Lowering the strength of promoter for expression of selective pressure resistance is an efficient strategy to increase selection stringency, which can be applied on industrial CHO-based cell line development platforms.

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures.

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2),the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d-glucose (2DG) and 5-thio- d-glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%-45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fctiters peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fctiters. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fcrevealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplementedcultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fcsialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%-33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.

Investigation and development of transient production process for porcine circovirus Type-2 (PCV2) capsid protein in HEK293F cells

Porcine circovirus type-2 capsid protein contains a major immunodominant epitope used as a subunit vaccine. Transient expression in mammalian cells is an efficient process for producing recombinant proteins. However, there is still a lack of research on the efficient production of virus capsid proteins in mammalian cells. Here we present a comprehensive study to investigate and optimize the production process of a model “difficult-to-express” virus capsid protein, PCV2 capsid protein in HEK293F transient expression system. The study evaluated the transient expression of PCV2 capsid protein in the mammalian cell line HEK293F and investigated the subcellular distribution by confocal microscopy. In addition, the RNA sequencing (RNA-seq) was used to detect the differential expression of genes after cells transfected with pEGFP-N1-Capsid or empty vectors. The analysis revealed that the PCV2 capsid gene affected a panel of differential genes of HEK293F cells involved in protein folding, stress response, and translation process, such as SHP90β, GRP78, HSP47, and eIF4A. An integrated strategy of protein engineering combined with VPA addition was applied to promote the expression of PCV2 capsid protein in HEK293F. Moreover, this study significantly increased the production of the engineered PCV2 capsid protein in HEK293F cells, reaching a yield of 8.7 mg/L. Conclusively, this study may provide deep insight for other “difficult-to-express” virus capsid proteins in the mammalian cell system.

CHO synthetic promoters improve expression and product quality of biotherapeutic proteins.

When expressing complex biotherapeutic proteins, traditional expression plasmids and methods may not always yield sufficient levels of high-quality product. High-strength viral promoters commonly used for recombinant protein (rProtein) production in mammalian cells allow for maximal expression, but provide limited scope to alter their transcription dynamics. However, synthetic promoters designed to provide tunable transcriptional activity offer a plasmid engineering approach to more precisely regulate product quality, yield or to reduce product related contaminants. We substituted the viral promoter CMV with synthetic promoters that offer different transcriptional activities to express our gene of interest in Chinese hamster ovary (CHO) cells. Stable pools were established and the benefits of regulating transgene transcription on the quality of biotherapeutics were examined in stable pool fed-batch overgrow experiments. Specific control of gene expression of the heavy chain (HC):light chain (LC) of a Fab, and the ratio between the two HCs in a Duet mAb reduced levels of aberrant protein contaminants; and the controlled expression of the helper gene XBP-1s improved expression of a difficult-to-express mAb. This synthetic promoter technology benefits applications that require custom activity. Our work highlights the advantages of employing synthetic promoters for production of more complex rProteins.

M echanism of lncRNA FOXD3 AS1 targeting miR 338 3p in human lens epithelial cell injury in diabetic cataract

Purpose: To explore the influence of lncRNA FOXD3-AS1 on high glucose (HG)-stimulated human lens epithelial cell injury in diabetic cataract. Methods: Lens epithelial cell HLEB3 were cultured in vitro and transfected with si-FOXD3-AS1, miR-338-3p mimic or miR-338-3p inhibitor, followed by HG (40 mmol/L) treatment. FOXD3-AS1, miR-338-3p, cleaved caspases3 and cleaved caspases9 expression were analyzed by RT-qPCR or western blot. SOD and CAT activities and MDA production in cells were determined using special kits. Cell apoptotic rate was quantified by flow cytometry. Regulatory relationship of FOXD3-AS1 and miR-338-3p was investigated by mechanism assay. Results: HG enhanced FOXD3-AS1 expression but repressed miR-338-3p (P&lt;0.05) in HLEB3 cells. FoxD3-AS1 depletion or miR-338-3p introduction promoted SOD and CAT activities, decreased MDA content, apoptosis rate, and cleaved caspases3 and cleaved caspases9 protein production in HG-induced HLEB3 cells (P&lt;0.05). FOXD3-AS1 could target and negatively regulate miR-338-3p. FOXD3-AS1-mediated influence on HG-induced HLEB3 cells was rescued by miR-338-3p inhibitors. Conclusion: FOXD3-AS1 silenced upregulated miR-338-3p expression to alleviate HG-induced oxidative stress and apoptosis of HLEB3 cells.

Optochemical Control of mRNA Translation in Eukaryotes.

A major stage in the expression of genes is the translation of messenger RNA (mRNA), and the regulation of this process is essential for protein production in cells. How tightly controlled gene expression can be spatially and temporally, is particularly evident in polar cells and embryonic development. We need tools to dissect these complex processes, if we wish to understand the underlying links, especially the difficulties brought on by malfunction. External bioorthogonal triggers are very helpful in this area, if they let us precisely control where and when a process is started. Equipping nucleic acids with light-responsive groups has proven to be an effective approach to examine the dynamic regulatory route of mRNA translation in living cells. In this review, we present an overview of the most recent methods for optochemically controlling translation, focusing on cis-acting technologies.

Optochemical Control of mRNA Translation in Eukaryotes

AbstractA major stage in the expression of genes is the translation of messenger RNA (mRNA), and the regulation of this process is essential for protein production in cells. How tightly controlled gene expression can be spatially and temporally, is particularly evident in polar cells and embryonic development. We need tools to dissect these complex processes, if we wish to understand the underlying links, especially the difficulties brought on by malfunction. External bioorthogonal triggers are very helpful in this area, if they let us precisely control where and when a process is started. Equipping nucleic acids with light‐responsive groups has proven to be an effective approach to examine the dynamic regulatory route of mRNA translation in living cells. In this review, we present an overview of the most recent methods for optochemically controlling translation, focusing on cis‐acting technologies.

Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome.

Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS.

Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier.

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries.