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2.
Biotechnol Bioeng ; 114(11): 2616-2627, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28734047

RESUMO

Site-specific recombinase mediated cassette exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines, this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here, we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers. The alternate start codon reduces the translation initiation efficiency and allows sorting for CHO cells with the highest transcription rates, while RMCE enables the subsequent exchange of the CD4 against the GOI. We have shown that sorted cell pools with the CD4 reporter gene containing the alternate start codon CTG lead to higher GFP signals and higher antibody titers upon RMCE as compared to cell pools containing the ATG start codon of the CD4 reporter. Despite the absence of any subcloning step, the final cell pool contained the CD4 gene in a single genome integration site.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Códon de Iniciação/genética , DNA Nucleotidiltransferases/genética , Técnicas de Transferência de Genes , Proteínas Recombinantes/genética , Ativação Transcricional/genética , Animais , Células CHO , Cricetinae , Cricetulus , Marcação de Genes/métodos , Engenharia de Proteínas/métodos , Transgenes/genética
3.
Biochem Pharmacol ; 120: 46-55, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27638414

RESUMO

The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Drosophila/metabolismo , Alucinógenos/metabolismo , N-Metil-3,4-Metilenodioxianfetamina/metabolismo , Serotoninérgicos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Alucinógenos/farmacologia , Humanos , Mutagênese Sítio-Dirigida , Mutação , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Serotonina/metabolismo , Serotoninérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Especificidade da Espécie , Especificidade por Substrato , Xenopus laevis
4.
Biotechnol Prog ; 31(2): 334-46, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25641927

RESUMO

N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production.


Assuntos
Engenharia Celular/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sialiltransferases/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Engenharia Metabólica , Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo
5.
J Biotechnol ; 193: 91-9, 2015 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-25444873

RESUMO

We report the discovery and validation of a novel CHO cell engineering target for improving IgG expression, serpin peptidase inhibitor, clade B, member 1 (Serpinb1). Transcriptomic studies using microarrays revealed that Serpinb1 was up-regulated in cultures with IgG heavy and light chain transcription transiently repressed compared with cultures treated with non-targeting siRNA. As proof of concept, a lentiviral vector was employed to overexpress the Chinese Hamster Serpinb1 in a CHOZN(®) Glutamine Synthetase (-/-) recombinant IgG producing CHO line. The lentiviral stable pool demonstrated 4.2-fold SERPINB1 overexpression compared with the non-transduced control. The peak viable cell density (VCD) and peak IgG volumetric productivity of the lentiviral stable pool increased 1.3 and 2.0 fold, respectively, compared with the non-transduced control. For host cell engineering, a plasmid encoding SERPINB1 was transfected into the CHOZN(®) GS (-/-) host cell line to create several stable pools. Single-cell clones isolated from the pools were characterized for their SERPINB1 expression levels and growth. The clone (SERPINB1_OE_27) with the highest SERPINB1 expression had decreased peak viable cell density and exponential phase growth rate. Selected SERPINB1 OE clones were subsequently evaluated for their IgG expression capabilities using GS selection. Clone SERPINB1_OE_42 with moderate SERPINB1 overexpression demonstrated increased IgG productivity in "bulk" selection. We conclude that manipulating Serpinb1 expression can lead to increased recombinant IgG productivity, but the effect in host cell lines may vary by clone and by overexpression level. This work represents the ongoing effort in applying "-omics" findings to novel CHO host cell line engineering.


Assuntos
Imunoglobulina G/metabolismo , Proteínas Recombinantes/metabolismo , Serpinas/metabolismo , Animais , Biotecnologia , Células CHO , Cricetinae , Cricetulus , Imunoglobulina G/análise , Imunoglobulina G/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Serpinas/química , Serpinas/genética
6.
Mol Cell Proteomics ; 13(12): 3224-35, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25092905

RESUMO

The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its "human-like" glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/).


Assuntos
Acetilgalactosamina/metabolismo , Eritropoetina/biossíntese , Glicoproteínas/química , N-Acetilgalactosaminiltransferases/metabolismo , Proteoma/química , Proteínas Recombinantes/biossíntese , Acetilgalactosamina/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Engenharia Celular , Linhagem Celular , Cricetulus , Eritropoetina/genética , Expressão Gênica , Glicômica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/genética , Proteoma/genética , Proteoma/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Alinhamento de Sequência , Polipeptídeo N-Acetilgalactosaminiltransferase
7.
J Biotechnol ; 167(1): 24-32, 2013 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-23777858

RESUMO

While complex N-linked glycoforms are often desired in biotherapeutic protein production, proteins with simple, homogeneous glycan structure have implications for X-ray crystallography and for recombinant therapeutics targeted to the mannose receptor of antigen presenting cells. Mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1, also called GnTI) adds N-acetylglucosamine to the Man5GlcNAc2 (Man5) N-glycan structure as part of complex N-glycan synthesis. Here, we report the use of zinc-finger nuclease (ZFN) genome editing technology to create Mgat1 disrupted Chinese hamster ovary (CHO) cell lines. These cell lines allow for the production of recombinant proteins with Man5 as the predominant N-linked glycosylation species. This method provides advantages over previously reported methods to create Mgat1-deficient cell lines. The use of ZFN-based genome editing eliminates potential regulatory concerns associated with random chemical mutagenesis, while retaining the robust growth and productivity characteristics of the parental cell lines. These Mgat1 disrupted cell lines may be used to produce mannose receptor-targeted therapeutic proteins. Cell line generation work can be performed in both Mgat1 disrupted and wild-type host cell lines to conduct X-ray crystallography studies of protein therapeutics in the same cell line used for production.


Assuntos
Endorribonucleases/genética , N-Acetilglucosaminiltransferases/genética , Fatores de Transcrição/genética , Animais , Células CHO , Cricetinae , Cricetulus , Técnicas de Inativação de Genes , Glutamato-Amônia Ligase/genética , Imunoglobulina G/metabolismo , Proteínas Recombinantes
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