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1.
Mol Cell ; 48(1): 39-51, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22981861

RESUMO

Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1ß, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.


Assuntos
Ácido Ascórbico/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Oxirredutases/metabolismo , Escorbuto/etiologia , Escorbuto/metabolismo , Animais , Ácido Ascórbico/farmacologia , Células Cultivadas , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/patologia , Modelos Animais de Doenças , Dissulfetos/metabolismo , Feminino , Glicoproteínas/deficiência , Glicoproteínas/genética , Masculino , Camundongos , Camundongos Mutantes , Mutação , Oxirredução , Oxirredutases/deficiência , Oxirredutases/genética , Peroxirredoxinas/deficiência , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Pró-Colágeno/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Escorbuto/genética , Escorbuto/patologia , Ácidos Sulfênicos/metabolismo , Fator de Crescimento Transformador beta/metabolismo
2.
Metab Eng ; 52: 143-152, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30513349

RESUMO

Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.


Assuntos
Células CHO/metabolismo , Proteína Inibidora do Complemento C1/biossíntese , Engenharia Metabólica/métodos , alfa 1-Antitripsina/biossíntese , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Sistemas CRISPR-Cas , Cricetinae , Cricetulus , Glicosilação , Humanos , Proteínas Recombinantes/biossíntese , Sialiltransferases/biossíntese , Sialiltransferases/genética
3.
Biotechnol Bioeng ; 111(8): 1604-16, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24827782

RESUMO

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named "CRISPy" for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells.


Assuntos
Células CHO/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fucosiltransferases/genética , Técnicas de Inativação de Genes/métodos , Chaperonas Moleculares/genética , Edição de RNA , Animais , Sequência de Bases , Cricetinae , Cricetulus , Endonucleases/genética , Endonucleases/metabolismo , Genoma , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Mutação INDEL , Internet , Dados de Sequência Molecular , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
4.
J Biol Chem ; 287(47): 39513-23, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-23027870

RESUMO

Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.


Assuntos
Estresse do Retículo Endoplasmático , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Resposta a Proteínas não Dobradas , Acetilcisteína/farmacologia , Substituição de Aminoácidos , Butionina Sulfoximina/farmacologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto , Oxirredução , Oxirredutases/genética
5.
Proteome Sci ; 8: 4, 2010 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-20205888

RESUMO

BACKGROUND: As part of a clinical proteomics program focused on diabetes and its complications we are looking for new and better protein biomarkers for diabetic nephropathy. The search for new and better biomarkers for diabetic nephropathy has, with a few exceptions, previously focused on either hypothesis-driven studies or urinary based investigations. To date only two studies have investigated the proteome of blood in search for new biomarkers, and these studies were conducted in sera from patients with type 2 diabetes. This is the first reported in depth proteomic study where plasma from type 1 diabetic patients was investigated with the goal of finding improved candidate biomarkers to predict diabetic nephropathy. In order to reach lower concentration proteins in plasma a pre-fractionation step, either hexapeptide bead-based libraries or anion exchange chromatography, was performed prior to surface enhanced laser desorption/ionization time-of-flight mass spectrometry analysis. RESULTS: Proteomic analysis of plasma from a cross-sectional cohort of 123 type 1 diabetic patients previously diagnosed as normoalbuminuric, microalbuminuric or macroalbuminuric, gave rise to 290 peaks clusters of which 16 were selected as the most promising biomarker candidates based on statistical performance, including independent component analysis. Four of the peaks that were discovered have been identified as transthyretin, apolipoprotein A1, apolipoprotein C1 and cystatin C. Several yet unidentified proteins discovered by this novel approach appear to have more potential as biomarkers for diabetic nephropathy. CONCLUSION: These results demonstrate the capacity of proteomic analysis of plasma, by confirming the presence of known biomarkers as well as revealing new biomarkers for diabetic nephropathy in plasma in type 1 diabetic patients.

6.
Sci Rep ; 9(1): 310, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670736

RESUMO

Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.


Assuntos
Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Animais , Células CHO , Cricetulus , Eritropoetina/metabolismo , Genes Reporter , Cinética , Proteínas Luminescentes/metabolismo , Espectrometria de Massas/métodos , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo
7.
ACS Synth Biol ; 8(4): 758-774, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30807689

RESUMO

Many branches of biology depend on stable and predictable recombinant gene expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined integration sites. However, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated expression cassette. Lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integration site and the expression cassette components. Here we show in a systematic manner how multiple components contribute to final net expression of recombinant genes in a characterized integration site. We develop a CRISPR/Cas9-based toolbox for construction of mammalian cell lines with targeted integration of a landing pad, containing a recombinant gene under defined 5' proximal regulatory elements. Generated site-specific recombinant cell lines can be used in a streamlined recombinase-mediated cassette exchange for fast screening of different expression cassettes. Using the developed toolbox, we show that different 5' proximal regulatory elements generate distinct and robust recombinant gene expression patterns in defined integration sites of CHO cells with a wide range of transcriptional outputs. This approach facilitates the generation of user-defined and product-specific gene expression patterns for programmable mammalian cell engineering.


Assuntos
Expressão Gênica/genética , Mamíferos/genética , Proteínas Recombinantes/genética , Animais , Células CHO , Sistemas CRISPR-Cas/genética , Engenharia Celular/métodos , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Cricetulus , Recombinases/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/genética
8.
Biotechnol J ; 13(3): e1700216, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29359860

RESUMO

The selection of clonally derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number are being used to assess suspension-adapted clones when grown statically in microtiter plates. However, no reported study so far has performed a direct head-to-head comparison of these two early reporters for predicting clone performance. Therefore, a screening platform for high-throughput analysis of titer and confluence of etanercept-producing clones is developed. Then an unbiased comparison of clone ranking based on either titer or titer normalized to confluence (TTC) is performed. Using two different suspension cultivation vessels, the authors demonstrate that titer- or TTC-based ranking gives rise to the selection of clones with similar volumetric productivity in batch cultures. Therefore, using both titer- and TTC-based ranking is proposed, allowing for selection of distinct clones with both high integral of viable cell density (IVCD) and high specific productivity features, respectively. This contributes to selection of a versatile panel of clones that can be further characterized and from which the final producer clone can be selected that best fits the production requirements.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Etanercepte/metabolismo , Glicoproteínas/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Contagem de Células , Cricetinae , Cricetulus , Etanercepte/química , Glicoproteínas/genética , Proteínas Recombinantes/genética
9.
Biotechnol Adv ; 35(1): 64-76, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27931938

RESUMO

Chinese hamster ovary (CHO) cells are the preferred cell factory for the production of therapeutic glycoproteins. Although efforts primarily within bioprocess optimization have led to increased product titers of recombinant proteins (r-proteins) expressed in CHO cells, post-transcriptional bottlenecks in the biosynthetic pathway of r-proteins remain to be solved. To this end, the ectopic expression of transgenes (effector genes) offers great engineering potential. However, studies on effector genes have in some cases led to inconsistent results. Whereas this can in part be attributed to product specificity, other experimental and cellular factors are likely important contributors to these conflicting results. Here, these factors are reviewed and discussed with the objective of guiding future studies on effector genes.


Assuntos
Células CHO , Engenharia Celular/métodos , Expressão Ectópica do Gene/genética , Proteínas Recombinantes , Animais , Células CHO/metabolismo , Cricetinae , Cricetulus , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Dosagem de Genes/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Biotechnol J ; 11(12): 1648-1656, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27731958

RESUMO

Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme-linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially available ELISA kits. We observed that estimations of recombinant human ø1-antitrypsin (rø1AT) titer by Coomassie-stained SDS-PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit. This prompted us to develop two independent quantification assays based on biolayer interferometry and reversed-phase high-performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off-the-shelf ELISA kits and found that the ELISA kits led to inconsistent results. The data presented here show that recombinant protein titers determined by ELISA kits cannot be trusted per se. Consequently, any ELISA kit to be used for determining recombinant protein titer must be validated by a different, preferably orthogonal method.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes/análise , alfa 1-Antitripsina/análise , Animais , Células CHO , Cromatografia Líquida de Alta Pressão/métodos , Cricetulus , Ensaio de Imunoadsorção Enzimática/normas , Glicosilação , Humanos , Interferometria/métodos , Kit de Reagentes para Diagnóstico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Padrões de Referência , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
11.
Sci Rep ; 5: 18016, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-26657798

RESUMO

Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells.


Assuntos
Técnicas de Cultura de Células , Ensaios de Triagem em Larga Escala , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Animais , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Genes Reporter , Glicosilação , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/química , Reprodutibilidade dos Testes
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