HEK293F Cells Research Articles

Relationships of Matrix Metalloproteinase 1 and a Tissue Inhibitor of Metalloproteinase to Collagen Metabolism in Haliotis discus hannai.

In response to physical, chemical, and/or biological stimuli, considerable tissue self-degradation occurs in abalone, causing severe post-harvest quality loss. During this process, the extracellular matrix (ECM) is greatly degraded by endogenous proteases. The main component of the ECM is collagen, primarily type I collagen. Although the activity of matrix metalloproteinases (MMPs), which can specifically degrade collagen, is precisely regulated by tissue inhibitors of MPs (TIMPs), indicating that MMPs and TIMPs play crucial roles in the regulation of tissue self-degradation, few studies have reported the interaction between MMPs and TIMPs. In this study, we reveal collagenases to participate in postmortem tissue self-degradation of Haliotis discus hannai by degrading type I collagen. The recombinant MMP-1 catalytic domain (rMMP1c) of abalone with high purity and enzyme activity is expressed using a prokaryotic expression system. The optimum temperature and pH for rMMP1c are 37 °C and 7.0, respectively. The thermal denaturation temperature of rMMP1c is 67.0 ± 0.9 °C. Ethylenediamine tetraacetic acid (EDTA) and 1,10-phenanthroline can completely inhibit rMMP1c activity, while Ba2+, Ca2+, and Mg2+ can significantly elevate it. TIMP is also expressed using HEK 293F cells. Recombinant TIMP (rTIMP) shows good inhibitory activity toward rMMP1c. Inhibition kinetics analyses reveal rTIMP to be a competitive inhibitor of rMMP1c. Biolayer interferometry reveals that rTIMP can effectively bind with rMMP1c, with an equilibrium dissociation constant value of 263 nM. rMMP1c effectively degrades type I collagen γ-β-α chains in turn, and rTIMP can significantly inhibit rMMP1c degradation activity. These results provide a theoretical basis for the study of MMP and TIMP interaction and elucidate the possible mechanism for abalone tissue self-degradation.

High-efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices.

Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here, we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X-ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.

Identification of three conserved linear B cell epitopes on the SARS-CoV-2 spike protein

ABSTRACT Spike (S) glycoprotein is the most significant structural protein of SARS-CoV-2 and a key target for neutralizing antibodies. In light of the on-going SARS-CoV-2 pandemic, identification and screening of epitopes of spike glycoproteins will provide vital progress in the development of sensitive and specific diagnostic tools. In the present study, NTD, RBD, and S2 genes were inserted into the pcDNA3.1(+) vector and designed with N-terminal 6× His-tag for fusion expression in HEK293F cells by transient transfection. Six monoclonal antibodies (4G, 9E, 4B, 7D, 8F, and 3D) were prepared using the expressed proteins by cell fusion technique. The characterization of mAbs was performed by indirect -ELISA, western blot, and IFA. We designed 49 overlapping synthesized peptides that cover the extracellular region of S protein in which 6 amino acid residues were offset between adjacent (S1–S49). Peptides S12, S19, and S49 were identified as the immunodominant epitope regions by the mAbs. These regions were further truncated and the peptides S12.2 286TDAVDCALDPLS297, S19.2 464FERDISTEIYQA475, and S49.4 1202ELGKYEQYIKWP1213 were identified as B- cell linear epitopes for the first time. Alanine scans showed that the D467, I468, E471, Q474, and A475 of the epitope S19.2 and K1205, Q1208, and Y1209 of the epitope S49.4 were the core sites involved in the mAbs binding. The multiple sequence alignment analysis showed that these three epitopes were highly conserved among the variants of concern (VOCs) and variants of interest (VOIs). Taken together, the findings provide a potential material for rapid diagnosis methods of COVID-19.

αvβ3-targeted sEVs for efficient intracellular delivery of proteins using MFG-E8

BackgroundSmall extracellular vesicles (sEVs) are nanometer-sized membranous particles shed by many types of cells and can transfer a multitude of cargos between cells. Recent studies of sEVs have been focusing on their potential to be novel drug carriers due to natural composition and other promising characteristics. However, there are challenges in sEVs-based drug delivery, one of which is the inefficient loading of drugs into sEVs, especially for large biomolecules.ResultsIn this study, we proposed a membrane-associated protein, milk fat globule–epidermal growth factor 8 protein (MFG-E8), to produce αvβ3-targeted sEVs with high delivery efficiency of interested protein. MFG-E8 is a secreted protein with NH2-terminal epidermal growth factor (EGF)–like domains, containing an Arg-Gly-Asp(RGD) sequence that binds αvβ3 and αvβ5 integrins, and COOH terminal domains C1 and C2, which can bind to lipid membrane with strong affinity. Firstly, we transiently expressed MFG-E8 in HEK293F cells and found that this protein could be secreted and adhere to the cell membrane. The recombinant MFG-E8 is also found to locate at the outer membrane of sEVs. Then we generated engineered sEVs by expressing high levels of the EGFP fused to MFG-E8 in HEK293F cells and showed that MFG-E8 could increase the delivery efficiency of EGFP into sEVs. Further delivery of Gaussia luciferase (GL) by fusion expression with MFG-E8 in donor cells demonstrated that target proteins fused with MFG-E8 still kept their activity. Finally, we identified the sEVs’ target to integrin αvβ3 by comparing the transfection efficiency with MFG-E8 loaded sEVs (MFG-E8-sEVs) in αvβ3 positive cells and αvβ3 negative cells. Analysis showed higher target protein could transfect into αvβ3 positive cells with MFG-E8-sEVs than with EGFP loaded sEVs (EGFP-sEVs), meaning the engineered sEVs with MFG-E8 not only could increase the delivery of target protein into sEVs, but also could target the αvβ3 positive cells.ConclusionThis study suggests that recombinant MFG-E8 is an ideal protein to increasingly deliver the drug into sEVs and give sEVs the ability to target the αvβ3 positive cells.

A novel multifunctional anti-PD-L1-CD16a-IL15 induces potent cancer cell killing in PD-L1-positive tumour cells

Cancer is the most acute disease and the leading cause of patient death worldwide. Both chemotherapy and molecular-based therapies play an important role in curing cancer. However, the median and overall survival of patients is poor. To date, immune therapies have changed the treatment methods for cancer patients. Programmed death ligand 1 (PD-L1, also known as B-H1, CD274) is a well-studied tumor antigen. PD-L1 is overexpressed in colon cancer, lung cancer, and so on and plays a vital role in cancer development. In this study, anti-PD-L1 single-domain antibodies were identified from recombinant human PD-L1 (rhPD-L1)-immunized llamas. Then, we generated a novel multifunctional anti-PD-L1-CD16a-IL15 antibody targeting PD-L1-positive tumor cells. Anti-PD-L1-CD16a-IL15 was constructed by linking the Interleukin-2 (IL-2) signal peptide, anti-PD-L1 single domain antibody (anti-PD-L1-VHH) and anti-cluster of differentiation 16a single domain antibody (anti-CD16a-VHH), and Interleukin-15/Interleukin-15 receptor alpha (IL15/IL-15Rα). This anti-PD-L1-CD16a-IL15 fusion protein can be expressed and purified from HEK-293F cells. In vitro, our data showed that the anti-PD-L1-CD16a-IL15 fusion protein can recruit T cells and drive natural killer cells (NK) with specific killing of PD-L1-overexpressing tumor cells. Furthermore, in the xenograft model, the anti-PD-L1-CD16a-IL15 fusion protein inhibited tumor growth with human peripheral blood mononuclear cells (PBMCs). These data suggested that the anti-PD-L1-CD16a-IL15 fusion protein has a latent function in antitumour activity, with better guidance for future cancer immunotherapy.

Development and characterization of recombinant ASFV CD2v protein nanoparticle-induced monoclonal antibody

African swine fever (ASF) caused by African swine fever virus (ASFV) is becoming a serious threat to the swine industry worldwide. CD2v is a key pathogenic factor of ASFV and the protective antigen with low immunogenicity, whereas viral protein-based nanoparticles have advantages of precise assembly and high immunogenicity. In this study, the CD2v protein fused with Norovirus (NoV) P particle assembled into nanoparticle for improved immunogenicity. Then, CD2v protein nanoparticle and monomer CD2v protein were expressed in HEK293F cells. The former induced higher levels of antibodies, and thus highly potent monoclonal antibodies (mAbs) were generated and characterized. The highest antibody titration of mAb 10A3 reached 1:2048000, and mAb 2E9 had the highest inhibition percent of 84% when competed with ASFV positive serum. Meanwhile, all mAbs reacted specifically with the denatured CD2v protein, and the linear epitope with the location of amino acids 28th to 51st of CD2v extracellular domain sequence was identified. In summary, this study produced a highly immunogenic CD2v protein and generated high-titer mAbs, the precise location of linear epitope on the CD2v was further determined. These findings may provide a powerful help for etiology and serological detection of ASFV.

Generation of Recombinant Antibodies in HEK293F Cells for the Detection of Staphylococcus aureus.

Staphylococcus aureus is a major resistant pathogen in clinical practice. Due to the increasing number of infections, rapid and sensitive detection of antibiotic-resistant S. aureus as well as antibiotic-sensitive S. aureus is important for the prevention and control of infectious diseases. In this study, we produced recombinant antibodies against S. aureus from mammalian human embryonic kidney 293 Freestyle cells with high yield and purity. These recombinant antibodies showed high binding affinity and low detection limit in both indirect and sandwich enzyme-linked immunosorbent assays for the detection of methicillin-resistant S. aureus and methicillin-sensitive S. aureus. These results suggest that the recombinant antibodies produced herein can be used for the accurate detection of S. aureus with a wild range of applications in medical diagnosis, food safety, and drug discovery.

Lysine-specific demethylase 1 as a corepressor of mineralocorticoid receptor.

Mineralocorticoid receptor (MR) and its ligand aldosterone play a central role in controlling blood pressure by promoting sodium reabsorption in the kidney. Coregulators are recruited to regulate the activation of steroid hormone receptors. In our previous study, we identified several new candidates for MR coregulators through liquid chromatography-tandem mass spectrometry analysis using a biochemical approach. Lysine-specific demethylase 1 (LSD1) was identified as a candidate. The relationship between LSD1 and salt-sensitive hypertension has been reported; however, the role of MR in this condition is largely unknown. Here, we investigated the functions of LSD1 as a coregulator of MR. First, a coimmunoprecipitation assay using HEK293F cells showed specific interactions between MR and LSD1. A chromatin immunoprecipitation study demonstrated LSD1 recruitment to the gene promoter of epithelial Na+ channel (ENaC), a target gene of MR. Reduced LSD1 expression by treatment with shRNA potentiated the hormonal activation of ENaC and serum/glucocorticoid-regulated kinase 1, another target gene of MR, indicating that LSD1 is a corepressor of MR. In an animal study, mice with kidney-specific LSD1 knockout (LSD1flox/floxKSP-Cre mice) developed hypertension after a high-salt diet without elevation of aldosterone levels, which was counteracted by cotreatment with spironolactone, an MR antagonist. In conclusion, our in vitro and in vivo studies demonstrated that LSD1 is a newly identified corepressor of MR.

A Transient Expression System with Stably Integrated CRISPR-dCas9 Fusions for Regulation of Genes Involved in Immunoglobulin G Glycosylation.

Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. Our understanding of molecular mechanisms that regulate IgG glycosylation is vague because of the complexity of this process, which involves hundreds of genes. Several genome-wide association (GWA) studies have revealed a network of genes associated with IgG glycosylation that are pleiotropic for a number of diseases. Here, we report a design of a versatile system for IgG production and gene manipulations that can be used for in vitro functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR-dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells. We validated our systems that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glyco-genes with a known role in IgG glycosylation, and then functionally validated three GWAS hits for IgG glycosylation with an as-yet-unknown role in this process.

Substitution of Pro165 in Transmembrane 4 of PfCRT Abolishes Lysosome Acidification Function in Stably Transfected HEK-293F Cells

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is localised on the parasite digestive vacuole, an organelle that maintains an acidic lumen. Here we demonstrated the isolation of HEK-293F cells stably expressing wild type 3D7 and mutant Pfcrt alleles. Immuno-fluorescence staining of HEK-293F transfectants confirms the localization of Pfcrt alleles to the lysosomal vesicles. Moreover, cells expressing mut-PfCRTETSE showed greater lysosomal acidification as demonstrated by the dramatic increase in the accumulation of two weak bases, acridine orange and CytiPainter LysoOrange dyes. Furthermore, HEK-293 cells stably expressing mut-PfCRTETSE with a single substitution of proline 165 in transmembrane 4 completely inhibited the accumulation of weak bases. Taken together, our results demonstrate the role of Pro165 in PfCRT lysosomal acidification function in HEK-293F cells.

High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells.

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein–protein or protein–membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins.

Purification of active human vacuolar H+-ATPase in native lipid-containing nanodiscs

Vacuolar H+-ATPases (V-ATPases) are large, multisubunit proton pumps that acidify the lumen of organelles in virtually every eukaryotic cell and in specialized acid-secreting animal cells, the enzyme pumps protons into the extracellular space. In higher organisms, most of the subunits are expressed as multiple isoforms, with some enriched in specific compartments or tissues and others expressed ubiquitously. In mammals, subunit a is expressed as four isoforms (a1-4) that target the enzyme to distinct biological membranes. Mutations in a isoforms are known to give rise to tissue-specific disease, and some a isoforms are upregulated and mislocalized to the plasma membrane in invasive cancers. However, isoform complexity and low abundance greatly complicate purification of active human V-ATPase, a prerequisite for developing isoform-specific therapeutics. Here, we report the purification of an active human V-ATPase in native lipid nanodiscs from a cell line stably expressing affinity-tagged a isoform 4 (a4). We find that exogenous expression of this single subunit in HEK293F cells permits assembly of a functional V-ATPase by incorporation of endogenous subunits. The ATPase activity of the preparation is >95% sensitive to concanamycin A, indicating that the lipid nanodisc-reconstituted enzyme is functionally coupled. Moreover, this strategy permits purification of the enzyme’s isolated membrane subcomplex together with biosynthetic assembly factors coiled-coil domain–containing protein 115, transmembrane protein 199, and vacuolar H+-ATPase assembly integral membrane protein 21. Our work thus lays the groundwork for biochemical characterization of active human V-ATPase in an a subunit isoform-specific manner and establishes a platform for the study of the assembly and regulation of the human holoenzyme.

Development of an indirect ELISA for detecting swine acute diarrhoea syndrome coronavirus IgG antibodies based on a recombinant spike protein.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhoea in neonatal piglets, leading to significant economic losses to the swine industry. Currently, there are no suitable serological methods to assess the infection of SADS-CoV and effectiveness of vaccines, making an urgent need to exploit effective enzyme-linked immunosorbent assay (ELISA) to compensate for this deficiency. Here, a recombinant plasmid that expresses the spike (S) protein of SADS-CoV fused to the Fc domain of human IgG was constructed to generate recombinant baculovirus and expressed in HEK 293F cells. The S-Fc protein was purified with protein G Resin, which retained reactivity with anti-human Fc and anti-SADS-CoV antibodies. The S-Fc protein was then used to develop an indirect ELISA (S-iELISA) and the reaction conditions of S-iELISA were optimized. As a result, the cut-off value was determined as 0.3711 by analyzing OD450nm values of 40 SADS-CoV-negative sera confirmed by immunofluorescence assay (IFA) and western blot. The coefficient of variation (CV) of 6 SADS-CoV-positive sera within and between runs of S-iELISA were both less than 10%. The cross-reactivity assays demonstrated that S-iELISA was non-cross-reactive with other swine viruses' sera. Furthermore, the overall coincidence rate between IFA and S-iELISA was 97.3% based on testing 111 clinical serum samples. Virus neutralization test with seven different OD450nm values of the sera showed that the OD450nm values tested by S-iELISA are positively correlated with the virus neutralization assay. Finally, a total of 300 pig field serum samples were tested by S-iELISA and commercial kits of other swine enteroviruses showed that the IgG-positive for SADS-CoV, TGEV, PDCoV and PEDV was 81.7, 54, 65.3 and 6%, respectively. The results suggest that this S-iELISA is specific, sensitive, repeatable and can be applied for the detection of the SADS-CoV infection in the swine industry.

IL4-10 Fusion Protein Shows DMOAD Activity in a Rat Osteoarthritis Model.

ObjectiveIdeally, disease-modifying osteoarthritis (OA) drugs (DMOAD) should combine chondroprotective, anti-inflammatory, and analgesic effects in a single molecule. A fusion protein of interleukin-4 (IL-4) and IL-10 (IL4-10 FP) possesses these combined effects. In this study, the DMOAD activity of rat IL4-10 FP (rIL4-10 FP) was tested in a rat model of surgically induced OA under metabolic dysregulation.DesignrIL4-10 FP was produced with HEK293F cells. Bioactivity of purified rIL4-10 FP was determined in a whole blood assay. Male Wistar rats (n = 20) were fed a high-fat diet (HFD) to induce metabolic dysregulation. After 12 weeks, OA was induced according to the Groove model. Two weeks after OA induction, rats were randomly divided into 2 groups and treated with 10 weekly, intra-articular injections of either rIL4-10 FP (n = 10) or phosphate buffered saline (PBS; n = 10). Possible antibody formation was evaluated using ELISA, cartilage degeneration and synovial inflammation were evaluated by histology and mechanical allodynia was evaluated using the von Frey test.ResultsIntra-articular injections with rIL4-10 FP significantly reduced cartilage degeneration (P = 0.042) and decreased mechanical allodynia (P < 0.001) compared with PBS. Only mild synovial inflammation was found (nonsignificant), limiting detection of putative anti-inflammatory effects. Multiple injections of rIL4-10 FP did not induce antibodies against rIL4-10 FP.ConclusionrIL4-10 FP showed chondroprotective and analgesic activity in a rat OA model with moderate cartilage damage, mild synovial inflammation, and pain. Future studies will need to address whether less frequent intra-articular injections, for example, with formulations with increased residence time, would also lead to DMOAD activity.

Functional recombinant human complement C1q with different affinity tags

Complement C1q is a multifunctional protein able to sense pathogens and immune molecules such as immunoglobulins and pentraxins, and to trigger the classical complement pathway through activation of its two associated proteases, C1r and C1s. C1q is a multimeric protein composed of three homologous yet distinct polypeptide chains A, B, and C, each composed of an N-terminal collagen-like sequence and a C-terminal globular gC1q module, that assemble into six heterotrimeric (A-B-C) subunits. This hexameric structure exhibits the characteristic shape of a bouquet of flowers, comprising six collagen-like triple helices, each terminating in a trimeric C-terminal globular head. We have produced previously functional recombinant full-length C1q in stably transfected HEK 293-F cells, with a FLAG tag inserted at the C-terminal end of C1qC chain. We report here the generation of additional recombinant C1q proteins, with a FLAG tag fused to the C-terminus of C1qA or C1qB chains, or to the N-terminus of the C1qC chain. Two other variants harboring a Myc or a 6-His tag at the C-terminal end of C1qC were also produced. We show that all C1q variants, except for the His-tagged protein, can be produced at comparable yields and are able to bind with similar affinities to either IgM, a ligand of the globular regions, or to the C1r2-C1s2 tetramer, and to trigger IgM-mediated serum complement activation. These new recombinant C1q variants provide additional tools to investigate the multiple functions of C1q.

Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells

Many therapeutic monoclonal antibodies require binding to Fc γ receptors (FcγRs) for full effect and increasing the binding affinity increases efficacy. Preeminent among the five activating human FcγRs is FcγRIIIa/CD16a expressed by natural killer (NK) cells. CD16a is heavily processed, and recent reports indicate that the composition of the five CD16a asparagine(N)-linked carbohydrates (glycans) impacts affinity. These observations indicate that specific manipulation of CD16a N-glycan composition in CD16a-expressing effector cells including NK cells may improve treatment efficacy. However, it is unclear if modifying the expression of select genes that encode processing enzymes in CD16a-expressing effector cells is sufficient to affect N-glycan composition. We identified substantial processing differences using a glycoproteomics approach by comparing CD16a isolated from two NK cell lines, NK92 and YTS, with CD16a expressed by HEK293F cells and previous reports of CD16a from primary NK cells. Gene expression profiling by RNA-Seq and qRT-PCR revealed expression levels for glycan-modifying genes that correlated with CD16a glycan composition. These results identified a high degree of variability between the processing of the same human protein by different human cell types. N-glycan processing correlated with the expression of glycan-modifying genes and thus explained the substantial differences in CD16a processing by NK cells of different origins.

Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies

Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins is challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our recent publication ( Liu et al., 2019 ). The procedure may be adapted for functional and structural studies.

The CAG promoter maintains high-level transgene expression in HEK293 cells.

The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lines, such as Chinese hamster ovary (CHO) cells, are decorated with human-like glycan structures that differ from those of human cells, and these may induce immunogenic responses in human cells. Human embryonic kidney cells (HEK293F) are also extensively used as hosts for the expression of recombinant therapeutic proteins, but their utility is limited by the low expression of transgenes in these cells. Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The expression levels and stability of the transgenes were evaluated by flow cytometry and qRT-PCR. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate-early enhancer combined with the chicken beta-actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33°C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta-actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expression. Although the temperature sensitivity of the CMV promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. When eGFP was replaced with hepatitis B surface antigen, the CAG promoter still showed the highest transgene expression. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant protein expression in HEK293F cells.

Sustained TNF-α stimulation leads to transcriptional memory that greatly enhances signal sensitivity and robustness.

Transcriptional memory allows certain genes to respond to previously experienced signals more robustly. However, whether and how the key proinflammatory cytokine TNF-α mediates transcriptional memory are poorly understood. Using HEK293F cells as a model system, we report that sustained TNF-α stimulation induces transcriptional memory dependent on TET enzymes. The hypomethylated status of transcriptional regulatory regions can be inherited, facilitating NF-κB binding and more robust subsequent activation. A high initial methylation level and CpG density around κB sites are correlated with the functional potential of transcriptional memory modules. Interestingly, the CALCB gene, encoding the proven migraine therapeutic target CGRP, exhibits the best transcriptional memory. A neighboring primate-specific endogenous retrovirus stimulates more rapid, more strong, and at least 100-fold more sensitive CALCB induction in subsequent TNF-α stimulation. Our study reveals that TNF-α-mediated transcriptional memory is governed by active DNA demethylation and greatly sensitizes memory genes to much lower doses of inflammatory cues.

Transcriptome Analysis of EPO and GFP HEK293 Cell-Lines Reveal Shifts in Energy and ER Capacity Support Improved Erythropoietin Production in HEK293F Cells

Higher eukaryotic cell lines like HEK293 are the preferred hosts for production of therapeutic proteins requiring human post translational processing. However, recombinant protein production can result in severe stress on the cellular machinery, resulting in limited titre and product quality. To investigate the cellular and metabolic characteristics associated with these limitations, we compared erythropoietin (secretory) and GFP (non-secretory) protein producer HEK293 cell-lines using transcriptomics analysis. Despite the high demand for ATP in all protein producer clones, a significantly higher capacity for ATP production was observed with erythropoietin producers as evidenced by the enrichment of upregulated genes in the oxidative phosphorylation pathway. In addition, ribosomal genes exhibited specific patterns of expression depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased erythropoietin secretion, we detected higher ER stress, including upregulation of the ATF6B gene. Our results are significant in recognizing key pathways for recombinant protein production and identifying potential target genes for further development of secretory power in mammalian cell factories.