RESUMO
With an increasing number of blockbuster drugs being recombinant mammalian proteins, protein production platforms that focus on mammalian proteins have had a profound impact in many areas of basic and applied research. Many groups, both academic and industrial, have been focusing on developing cost-effective methods to improve the production of mammalian proteins that would support potential therapeutic applications. As it stands, while a wide range of platforms have been successfully developed for laboratory use, the majority of biologicals are still produced in mammalian cell lines due to the requirement for posttranslational modification and the biosynthetic complexity of target proteins. An unbiased high-throughput RNAi screening approach can be an efficient tool to identify target genes involved in recombinant protein production. Here, we describe the process of optimizing the transfection conditions, performing the genome-wide siRNA screen, the activity and cell viability assays, and the validation transfection to identify genes involved with protein expression.
Assuntos
Ensaios de Triagem em Larga Escala , Interferência de RNA , RNA Interferente Pequeno , Transfecção , Ensaios de Triagem em Larga Escala/métodos , Humanos , RNA Interferente Pequeno/genética , Transfecção/métodos , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sobrevivência Celular/genéticaRESUMO
Placental malaria vaccines (PMVs) are being developed to prevent severe sequelae of placental malaria (PM) in pregnant women and their offspring. The leading candidate vaccine antigen VAR2CSA mediates parasite binding to placental receptor chondroitin sulfate A (CSA). Despite promising results in small animal studies, recent human trials of the first two PMV candidates (PAMVAC and PRIMVAC) generated limited cross-reactivity and cross-inhibitory activity to heterologous parasites. Here we immunized Aotus nancymaae monkeys with three PMV candidates (PAMVAC, PRIMVAC and ID1-ID2a_M1010) adjuvanted with Alhydrogel, and exploited the model to investigate boosting of functional vaccine responses during PM episodes as well as with nanoparticle antigens. PMV candidates induced high levels of antigen-specific IgG with significant cross-reactivity across PMV antigens by enzyme-linked immunosorbent assay. Conversely, PMV antibodies recognized native VAR2CSA and blocked CSA adhesion of only homologous parasites and not of heterologous parasites. PM episodes did not significantly boost VAR2CSA antibody levels or serum functional activity; nanoparticle and monomer antigens alike boosted serum reactivity but not functional activities. Overall, PMV candidates induced functional antibodies with limited heterologous activity in Aotus monkeys, similar to responses reported in humans. The Aotus model appears suitable for preclinical downselection of PMV candidates and assessment of antibody boosting by PM episodes.
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária , Animais , Humanos , Feminino , Gravidez , Placenta/parasitologia , Malária Falciparum/prevenção & controle , Malária Falciparum/parasitologia , Plasmodium falciparum , Antígenos de Protozoários , Anticorpos Antiprotozoários , Malária/prevenção & controle , Aotidae , ImunidadeRESUMO
Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.
Assuntos
Acetato-CoA Ligase , Escherichia coli , Acetato Quinase/genética , Acetato Quinase/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Escherichia coli/metabolismo , Óperon , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismoRESUMO
Aerobic Escherichia coli growth at restricted iron concentrations (≤ 1.75 ± 0.04 µM) is characterized by lower biomass yield, higher acetate accumulation and higher activation of the siderophore iron-acquisition systems. Although iron homeostasis in E. coli has been studied intensively, previous studies focused only on understanding the regulation of the iron import systems and the iron-requiring enzymes. Here, the effect of iron availability on the energy metabolism of E. coli has been investigated. It was established that aerobic cultures growing under limiting iron conditions showed lower ATP yield per glucose, lower growth rate and lower TCA cycle activity and respiration, at the same time as increased glucose consumption, acetate and pyruvate accumulation, practically mimicking microaerobic growth. However, at excess iron, independent of oxygen availability, the cultures showed high cellular energetics (5.8 ATP/mol of glucose) by using pathways requiring iron-rich complex proteins found in the TCA cycle and respiratory chain. In conditions of iron excess, some iron-requiring terminal reductases of the respiratory chain, that were thought to function only under anaerobiosis, were used by the E. coli, when in aerobic conditions, to maintain high respiratory activity. This allowed it to produce more biomass and more reactive oxygen species that were controlled by the higher activity of the antioxidant defenses (SOD, peroxidase and catalase) and the iron-sulfur cluster repair systems.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Trifosfato de Adenosina , Anaerobiose , Transporte de Elétrons , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Ferro/metabolismo , Oxirredutases/metabolismoRESUMO
Human SERPINF1 gene codes for pigment epithelium-derived factor (PEDF), a secreted glycoprotein and member of the SERPIN superfamily. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. The cells produced and secreted recombinant PEDF proteins into the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. PEDF has affinity for PEDF-receptor (PEDF-R), a membrane-linked lipase encoded by the PNPLA2 gene. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3'end deletions and by induction with isopropyl ß-d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. C-terminally truncated PEDF-R versions containing the ligand binding region retained the ability to bind PEDF. The data demonstrate that mammalian-derived recombinant PEDF and bacterially derived recombinant PEDF-R can be produced and purified in large amounts for further use in structural and biological studies.
Assuntos
Serpinas , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Mamíferos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Receptores de Neuropeptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serpinas/genética , Serpinas/metabolismoRESUMO
In the literature, there are few high-throughput screens or even methods for high-throughput screens of recombinant adeno-associated virus (rAAV) production despite potential benefits to research and production. In this study, a generalizable high-throughput relative rAAV titration method is examined within the context of an siRNA screen as siRNA knockdown is a common means of pathway engineering in bioproduction. Crude samples generated from transfected HEK293T/17 cultures were subjected to quantitative PCR (qPCR) and used to transduce COS7 cells to assess relative differences in genomic and infectious rAAV titer, respectively, at the 384-well scale, evaluating both supernatant and lysed samples. To evaluate relevant differences in titer for conditions that could be used in an actual screen, cultures subjected to an siRNA reverse transfection and subsequent rAAV forward transfection were also tested. The delayed forward rAAV triple-plasmid transfection was not seen to affect the siRNA activity of tested controls, while siRNA transfection was shown to measurably impact rAAV titer. Effective differentiation between infectious titer levels was dependent upon the choice of sample dilution, but trends between qPCR and infectious titer assays were consistent across sample sets.
Assuntos
Dependovirus , Vetores Genéticos , Dependovirus/genética , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , TransfecçãoRESUMO
The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line's most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.
Assuntos
Engenharia Celular/métodos , Proliferação de Células/genética , Edição de Genes/métodos , Transgenes , Sistemas CRISPR-Cas , Adesão Celular , Células Clonais , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , TransfecçãoRESUMO
Granulibacter bethesdensis can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact G. bethesdensis (Gb) is hypostimulatory compared to Escherichia coli, i.e., cytokine production in human blood requires 10-100 times more G. bethesdensis CFU/mL than E. coli. To better understand the pathogenicity of G. bethesdensis, we isolated its lipopolysaccharide (GbLPS) and characterized its lipid A. Unlike with typical Enterobacteriaceae, the release of presumptive Gb lipid A from its LPS required a strong acid. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of α-Manp-(1â4)-ß-GlcpN3N-(1â6)-α-GlcpN-(1â¿1)-α-GlcpA tetra-saccharide substituted with five acyl chains: the amide-linked N-3' 14:0(3-OH), N-2' 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constituent of the core region of the LPS that is covalently attached to GlcpN3N of the lipid backbone may account for the acid resistance of GbLPS. In addition, the presence of Ko and only five acyl chains may explain the >10-fold lower proinflammatory potency of GbKo-lipidA compared to E. coli lipid A, as measured by cytokine induction in human blood. These unusual structural properties of the G.bethesdensis Ko-lipid A glycolipid likely contribute to immune evasion during pathogenesis and resistance to antimicrobial peptides.
Assuntos
Acetobacteraceae/metabolismo , Doença Granulomatosa Crônica/microbiologia , Lipídeo A/química , Acetatos/análise , Acetobacteraceae/isolamento & purificação , Acetobacteraceae/patogenicidade , Sequência de Carboidratos , Citocinas/sangue , Doença Granulomatosa Crônica/sangue , Humanos , Lipídeo A/metabolismoRESUMO
The signal that triggers the expression of the ace operon and, in turn, the transition of central metabolism's architecture from acetogenic to gluconeogenic in Escherichia coli remains elusive despite extensive research both in vivo and in vitro. Here, with the aid of flux analysis together with measurements of the enzymic activity of isocitrate lyase (ICL) and its aceA-messenger ribonucleuc acid (mRNA) transcripts, we provide credible evidence suggesting that the expression of the ace operon in E. coli is triggered in response to growth rate-dependent threshold flux-signal of adenosine triphosphate (ATP). Flux analysis revealed that the shortfall in ATP supply observed as the growth rate ($\mu $) diminishes from µmax to ≤ 0.43h-1 ($ \pm 0.02;n4)\ $is partially redressed by up-regulating flux through succinyl CoA synthetase. Unlike glycerol and glucose, pyruvate cannot feed directly into the two glycolytic ATP-generating reactions catalyzed by phosphoglycerokinase and pyruvate kinase. On the other hand, glycerol, which upon its conversion to D-glyceraldehyde, feeds into the phosphorylation and dephosphorylation parts of glycolysis including the substrate-level phosphorylation-ATP generating reactions, thus preventing ATP flux from dropping to the critical threshold signal required to trigger the acetate-diauxic switch until glycerol is fully consumed. The mRNA transcriptional patterns of key gluconeogenic enzymes, namely, ackA, acetate kinase; pta, phosphotransacetylase; acs, acetyl CoA synthetase and aceA, ICL, suggest that the pyruvate phenotype is better equipped than the glycerol phenotype for the switch from acetogenic to gluconeogenic metabolism.
Assuntos
Trifosfato de Adenosina/metabolismo , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome-wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line.
Assuntos
Proteínas Reguladoras de Apoptose/deficiência , Proteínas de Ligação ao Cálcio/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação para Cima , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: Lethal B. anthracis infection produces high proinflammatory peptidoglycan (PGN) burdens in hosts. We investigated whether the lethality and inflammation anthrax PGN can produce are related. METHODS: At 6 h before and the start of 24 h anthrax PGN infusions, rats (n = 198) were treated with diluent (controls) or one of three IV-doses of either hydrocortisone (125, 12.5 or 1.25 mg/kg) or TNF-soluble receptor (TNFsr; 2000, 1000 or 333 µg/kg), non-selective and selective anti-inflammatory agents, respectively. RESULTS: Compared to controls, hydrocortisone 125 and 12.5 mg/kg each decreased 7-day lethality (p ≤ 0.004). Hydrocortisone 125 mg/kg decreased IL-1ß, IL-6, TNFα, MCP, MIP-1α, MIP-2, RANTES and nitric oxide (NO) blood levels at 4 and 24 h after starting PGN (except MCP at 24 h). Each decrease was significant at 4 h (except MIP-1α that was significant at 24 h) (p ≤ 0.05). Similarly, hydrocortisone 12.5 mg/kg decreased each measure at 4, 24 and 48 h (except TNFα at 24 h and MIP-1α at 24 and 48 h and NO at 48 h). Decreases were significant for IL-6 and NO at 4 h and RANTES at 48 h (p ≤ 0.05). Hydrocortisone 1.25 mg/kg had non-significant effects. Each TNFsr dose decreased lethality but non-significantly. However, when doses were analyzed together, TNFsr decreased lethality in a potential trend (p = 0.16) and IL-6 and NO significantly at 4 h (p = 0.05). CONCLUSIONS: Peptidoglycan-stimulated host inflammation may contribute to B. anthracis lethality.
RESUMO
BACKGROUND: The expression of recombinant proteins triggers a stress response which downregulates key metabolic pathway genes leading to a decline in cellular health and feedback inhibition of both growth and protein expression. Instead of individually upregulating these downregulated genes or improving transcription rates by better vector design, an innovative strategy would be to block this stress response thereby ensuring a sustained level of protein expression. RESULTS: We postulated that the genes which are commonly up-regulated post induction may play the role of signalling messengers in mounting the cellular stress response. We identified those genes which have no known downstream regulatees and created knock outs which were then tested for GFP expression. Many of these knock outs showed significantly higher expression levels which was also sustained for longer periods. The highest product yield (Yp/x) was observed in a BW25113ΔcysJ knock out (Yp/x 0.57) and BW25113ΔelaA (Yp/x 0.49), whereas the Yp/x of the control W3110 strain was 0.08 and BW25113 was 0.16. Double knock out combinations were then created from the ten best performing single knock outs leading to a further enhancement in expression levels. Out of 45 double knock outs created, BW25113ΔelaAΔyhbC (Yp/x 0.7) and BW25113ΔcysJΔyhbC (Yp/x 0.64) showed the highest increase in product yield compared to the single gene mutant strains. We confirmed the improved performance of these knock outs by testing and obtaining higher levels of recombinant asparaginase expression, a system better suited for analysing sustained expression since it gets exported to the extracellular medium. CONCLUSION: Creating key knock outs to block the CSR and enhance expression is a radically different strategy that can be synergistically combined with traditional methods of improving protein yields thus helping in the design of superior host platforms for protein expression.
Assuntos
Asparaginase/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Inativação de Genes/métodos , Asparaginase/genética , Proteínas de Escherichia coli/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas de Fluorescência Verde/biossíntese , Redes e Vias Metabólicas/genética , Proteínas Recombinantes/biossíntese , Transdução de Sinais/genética , Estresse Fisiológico , Regulação para CimaRESUMO
GRTH/DDX25 is a testicular RNA helicase expressed in germ cells that plays a crucial role in completion of spermatogenesis. Previously, we demonstrated a missense mutation (R242H) of GRTH gene in Japanese infertile patients (5.8%) with non-obstructive azoospermia. This mutation upon expression in COS-1 cells revealed absence of the 61 kDa phosphorylated GRTH in cytoplasm and the presence of the 56 kDa non-phosphorylated GRTH in the nucleus. GRTH knock-in (KI) mice carrying the human GRTH (R242H) mutation, lack phosphorylated GRTH, and sperm due to failure of round spermatid elongation during spermiogenesis. To determine the impact of phosphorylated GRTH on molecular events/pathways participating in spermatid development during spermiogenesis, we analyzed transcriptome profiles obtained from RNA-Seq of germ cells from KI and WT mice. RNA-Seq analysis of 2624 differentially expressed genes revealed 1404 down-regulated and 1220 up-regulated genes in KI mice. Genes relevant to spermatogenesis, spermatid development and spermatid differentiation were significantly down-regulated. KEGG enrichment analysis showed genes related to ubiquitin-mediated proteolysis and protein processing in endoplasmic reticulum pathway genes were significantly down-regulated while the up-regulated genes were found to be involved in Focal adhesion and ECM-receptor interaction pathways. Real-Time PCR analysis confirmed considerable reduction in transcripts of ubiquitination related genes Ube2j1, Ube2k, Ube2w, Rnf8, Rnf133, Rnf138, Cul3 and increased expression of Ccnd2, Col1a, Lamb1, Cav1, Igf1, Itga9 mRNA's in KI mice compared to WT. Also, marked reduction in protein expression of UBE2J1, RNF8, RNF138 (ubiquitination network), MOF (histone acetyltransferase), their modified Histone substrates (H2AUb, H2BUb) and H4Ac, H4K16Ac were observed in KI mice. GRTH-IP mRNA binding studies revealed that Rnf8 and Ube2J1 mRNAs from WT mice associated with GRTH protein and the binding is greatly impaired in the KI mice. Immunohistochemistry analysis showed significantly reduced expression of RNF8, MOF, H4Ac and H4K16Ac in round spermatids of KI mice. Absence of phosphorylated GRTH impairs UBE2J1, RNF8 and MOF-dependent histone ubiquitination and acetylation essential for histone replacement, chromatin condensation and spermatid elongation during spermiogenesis.
RESUMO
The repeating unit of the C. difficile Toxin A (rARU, also known as CROPS [combined repetitive oligopeptides]) C-terminal region, was shown to elicit protective immunity against C. difficile and is under consideration as a possible vaccine against this pathogen. However, expression of recombinant rARU in E. coli using the standard vaccine production process was very low. Transcriptome and proteome analyses showed that at restricted dissolved oxygen (DO) the numbers of differentially expressed genes (DEGs) was 2.5-times lower than those expressed at unrestricted oxygen. Additionally, a 7.4-times smaller number of ribosome formation genes (needed for translation) were down-regulated as compared with unrestricted DO. Higher rARU expression at restricted DO was associated with up-regulation of 24 heat shock chaperones involved in protein folding and with the up-regulation of the global regulator RNA chaperone hfq. Cellular stress response leading to down-regulation of transcription, translation, and energy generating pathways at unrestricted DO were associated with lower rARU expression. Investigation of the C. difficile DNA sequence revealed the presence of cell wall binding profiles, which based on structural similarity prediction by BLASTp, can possibly interact with cellular proteins of E. coli such as the transcriptional repressor ulaR, and the ankyrins repeat proteins. At restricted DO, rARU mRNA was 5-fold higher and the protein expression 27-fold higher compared with unrestricted DO. The report shows a strategy for improved production of C. difficile vaccine candidate in E. coli by using restricted DO growth. This strategy could improve the expression of recombinant proteins from anaerobic origin or those with cell wall binding profiles.
Assuntos
Toxinas Bacterianas/genética , Enterotoxinas/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Oxigênio/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/química , Enterotoxinas/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Cinética , Modelos Biológicos , Subunidades Proteicas/química , Proteômica , SolubilidadeRESUMO
Stable cell lines can continuously produce a recombinant protein without the need to repeatedly engineer the genome. In a previous study HIPK1, Homeodomain-interacting Protein Kinase 1, was found to be a target of the microRNA miR-22 that, when repressed, improved expression of both an intracellular and a secreted protein. In this report, HEK293 cells stably over-expressing miR-22 were compared with HEK293 with knockout of HIPK1, executed by CRISPR/Cas9, for their ability to improve recombinant protein expression. In this model case of luciferase, over-expression of miR-22 improved overall activity 2.4-fold while the HIPK1 knockout improved overall activity 4.7-fold.
Assuntos
MicroRNAs/genética , Biossíntese de Proteínas/genética , Proteínas Serina-Treonina Quinases/genética , Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , HumanosRESUMO
High-throughput siRNA screens were only recently applied to cell factories to identify novel engineering targets which are able to boost cells towards desired phenotypes. While siRNA libraries exist for model organisms such as mice, no CHO-specific library is publicly available, hindering the application of this technique to CHO cells. The optimization of these cells is of special interest, as they are the main host for the production of therapeutic proteins. Here, we performed a cross-species approach by applying a mouse whole-genome siRNA library to CHO cells, optimized the protocol for suspension cultured cells, as this is the industrial practice for CHO cells, and developed an in silico method to identify functioning siRNAs, which also revealed the limitations of using cross-species libraries. With this method, we were able to identify several genes that, upon knockdown, enhanced the total productivity in the primary screen. A second screen validated two of these genes, Rad21 and Chd4, whose knockdown was tested in additional CHO cell lines, confirming the induced high productivity phenotype, but also demonstrating the cell line/clone specificity of engineering effects.
Assuntos
Biblioteca Gênica , Genoma/genética , Ensaios de Triagem em Larga Escala/métodos , RNA Interferente Pequeno/genética , Animais , Células CHO , Técnicas de Cultura de Células/métodos , Proteínas de Ciclo Celular/genética , Cricetinae , Cricetulus , DNA Helicases/genética , Perfilação da Expressão Gênica/métodos , Humanos , Interferência de RNARESUMO
Pichia pastoris KM71H (MutS ) is an efficient producer of hard-to-express proteins such as the membrane protein P-glycoprotein (Pgp), an ATP-powered efflux pump which is expressed properly, but at very low concentration, using the conventional induction strategy. Evaluation of different induction strategies indicated that it was possible to increase Pgp expression by inducing the culture with 20% media containing 2.5% methanol. By quantifying methanol, formaldehyde, hydrogen peroxide and formate, and by measuring alcohol oxidase, catalase, formaldehyde dehydrogenase, formate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases, it was possible to correlate Pgp expression to the induction strategy. Inducing the culture by adding methanol with fresh media was associated with decreases in formaldehyde and hydrogen peroxide, and increases in formaldehyde dehydrogenase, formate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases. At these conditions, Pgp expression was 1400-fold higher, an indication that Pgp expression is affected by increases in formaldehyde and hydrogen peroxide. It is possible that Pgp is responsible for this behaviour, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions. This report adds information on methanol metabolism during expression of Pgp from P. pastoris MutS strain and suggests an expression procedure for hard-to-express proteins from P. pastoris.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Expressão Gênica , Metanol/metabolismo , Pichia/enzimologia , Pichia/metabolismo , Ativação Transcricional/efeitos dos fármacos , Meios de Cultura/química , Perfilação da Expressão Gênica , Pichia/efeitos dos fármacosRESUMO
Growing E. coli to high densities is a common strategy for biologicals production. The process is implemented by using complex or minimal media with different feeding strategies. To understand the effect of amino acids, E. coli B and K were grown at a steady state of 0.35 h-1 in glucose minimal medium with and without amino acids, while their metabolism, protein abundance and gene expression were compared. The results showed that amino acids promoted higher acetate excretion, higher fatty acid biosynthesis (K strain), repressed glucose uptake rate, and decreased expression of proteins associated with the TCA cycle, glyoxylate shunt and amino acid biosynthesis. In presence of amino acids, E. coli K upregulated fatty acid biosynthesis and repressed more genes and proteins involved in amino acid biosynthesis than E. coli B. These findings are correlated with higher yield on glucose (Yx/s) and high specific biomass production rate (qx) in K strain in the presence of amino acids. In contrast, pre-formed precursor molecules such as amino acids did not affect fatty acid biosynthesis in E. coli B or Yx/s and qx, which were higher than those of E. coli K, suggesting that constitutive synthesis of energetically demanding precursors and higher fatty acid ß-oxidation activity is key for high biomass-performer E. coli B. Both strains turned off unnecessary pathways and directed their metabolism to proteome efficient overflow metabolism likely to generate energy and provide protein to functions supporting higher growth rate.
Assuntos
Aminoácidos/farmacologia , Meios de Cultura/farmacologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Genes Bacterianos , Biossíntese de Proteínas/genética , Transcrição Gênica/efeitos dos fármacos , Reatores Biológicos/microbiologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Biossíntese de Proteínas/efeitos dos fármacos , Proteômica , Transcriptoma/genéticaRESUMO
Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the systems tested introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is considered as a preferred method. The present study examines the effect of PA expression on the metabolism of B. anthracis producing strain, BH500, by comparing it with a control strain carrying an empty plasmid. The strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was conducted. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase σ54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. There were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Also, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain during the lag phase and down-regulated in the log and late-log phases, which was associated with decreased specific growth rates. The information obtained from this study may guide genetic modification of B. anthracis to improve PA production.