RESUMO
Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Eritropoetina/genética , Eritropoetina/metabolismo , Glutamato-Amônia Ligase/genética , Engenharia de Proteínas/métodos , Sistemas CRISPR-Cas , Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos/genética , Glicosilação , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Recombinantes/metabolismoRESUMO
CRISPR-Cas9 has emerged as a powerful technology that enables ready modification of the mammalian genome. The ability to modulate Cas9 activity can reduce off-target cleavage and facilitate precise genome engineering. Here we report the development of a Cas9 variant whose activity can be switched on and off in human cells with 4-hydroxytamoxifen (4-HT) by fusing the Cas9 enzyme with the hormone-binding domain of the estrogen receptor (ERT2). The final optimized variant, termed iCas, showed low endonuclease activity without 4-HT but high editing efficiency at multiple loci with the chemical. We also tuned the duration and concentration of 4-HT treatment to reduce off-target genome modification. Additionally, we benchmarked iCas against other chemical-inducible methods and found that it had the fastest on rate and that its activity could be toggled on and off repeatedly. Collectively, these results highlight the utility of iCas for rapid and reversible control of genome-editing function.
Assuntos
Sistemas CRISPR-Cas/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Tamoxifeno/análogos & derivados , Células HEK293 , Humanos , Tamoxifeno/química , Tamoxifeno/farmacologiaRESUMO
In yeast Saccharomyces cerevisiae, ~3% of the lysine transfer RNA acceptor 1 (tRK1) pool is imported into mitochondria while the second isoacceptor, tRK2, fully remains in the cytosol. The mitochondrial function of tRK1 is suggested to boost mitochondrial translation under stress conditions. Strikingly, yeast tRK1 can also be imported into human mitochondria in vivo, and can thus be potentially used as a vector to address RNAs with therapeutic anti-replicative capacity into mitochondria of sick cells. Better understanding of the targeting mechanism in yeast and human is thus critical. Mitochondrial import of tRK1 in yeast proceeds first through a drastic conformational rearrangement of tRK1 induced by enolase 2, which carries this freight to the mitochondrial pre-lysyl-tRNA synthetase (preMSK). The latter may cross the mitochondrial membranes to reach the matrix where imported tRK1 could be used by the mitochondrial translation apparatus. This work focuses on the characterization of the complex that tRK1 forms with human enolases and their role on the interaction between tRK1 and human pre-lysyl-tRNA synthetase (preKARS2).
Assuntos
Lisina-tRNA Ligase/metabolismo , Mitocôndrias/metabolismo , Fosfopiruvato Hidratase/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Algoritmos , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Proteínas de Transporte de Cátions/metabolismo , Citosol/metabolismo , Bases de Dados de Proteínas , Células Hep G2 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Mitochondrial import of small noncoding RNA is found in a large variety of species. In mammalian cells, this pathway can be used for therapeutic purpose, to restore the mitochondrial functions affected by pathogenic mutations. Recently, we developed mitochondrial RNA vectors able to address therapeutic oligoribonucleotides into human mitochondria. Here we provide the protocol for transfection of cultured human cells with small recombinant RNA molecules and describe two approaches useful to demonstrate their import into mitochondria: (1) isolation of RNA from purified mitochondria and quantitative hybridization analysis and (2) confocal microscopy of cells transfected with fluorescently labeled RNA. These protocols can be used in combination with overexpression or downregulation of protein import factors to detect and to evaluate their influence on the mitochondrial import of various RNAs.
Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA/genética , Células Cultivadas , Expressão Gênica , Humanos , Microscopia Confocal , Microscopia de Fluorescência , RNA/metabolismo , Transporte de RNA , RNA Mitocondrial , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNA(Lys)(CUU) into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNA(Lys)(CUU) and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms.
Assuntos
Lisina-tRNA Ligase/metabolismo , Mitocôndrias/enzimologia , RNA de Transferência de Lisina/metabolismo , RNA de Transferência/metabolismo , Sequência de Bases , Proteínas de Transporte de Cátions/química , Ensaio de Desvio de Mobilidade Eletroforética , Células Hep G2 , Humanos , Sequências Repetidas Invertidas , Lisina-tRNA Ligase/química , Mitocôndrias/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Transporte de RNA , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA de Transferência de Lisina/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Multiple-respiratory-chain deficiency represents an important cause of mitochondrial disorders. Hitherto, however, mutations in genes involved in mtDNA maintenance and translation machinery only account for a fraction of cases. Exome sequencing in two siblings, born to consanguineous parents, with severe encephalomyopathy, choreoathetotic movements, and combined respiratory-chain defects allowed us to identify a homozygous PNPT1 missense mutation (c.1160A>G) that encodes the mitochondrial polynucleotide phosphorylase (PNPase). Blue-native polyacrylamide gel electrophoresis showed that no PNPase complex could be detected in subject fibroblasts, confirming that the substitution encoded by c.1160A>G disrupts the trimerization of the protein. PNPase is predominantly localized in the mitochondrial intermembrane space and is implicated in RNA targeting to human mitochondria. Mammalian mitochondria import several small noncoding nuclear RNAs (5S rRNA, MRP RNA, some tRNAs, and miRNAs). By RNA hybridization experiments, we observed a significant decrease in 5S rRNA and MRP-related RNA import into mitochondria in fibroblasts of affected subject 1. Moreover, we found a reproducible decrease in the rate of mitochondrial translation in her fibroblasts. Finally, overexpression of the wild-type PNPT1 cDNA in fibroblasts of subject 1 induced an increase in 5S rRNA import in mitochondria and rescued the mitochondrial-translation deficiency. In conclusion, we report here abnormal RNA import into mitochondria as a cause of respiratory-chain deficiency.