Novel CNBP‐ and La‐based translation control systems for mammalian cells

Schlatter, Stefan; Fussenegger, Martin

doi:10.1002/bit.10549

Cited by 31 publications

(25 citation statements)

References 70 publications

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“…Different metabolic engineering strategies have been designed since to reduce production bottlenecks and improve the specific productivity of mammalian host cell lines or the quality of the product. Most successful examples include (i) transcription engineering using trigger-inducible expression systems (Schlatter and Fussenegger, 2003), (ii) translation engineering consisting of improving ribosomal entry and translationinitiation (Underhill et al, 2003;Umaña et al, 1999), (iii) glycoengineering improving the ADCC activity of therapeutic antibodies (Prati et al, 2002), (iv) controlled proliferation technology which redirects metabolic energy from cell growth to product formation (Fussenegger et al, 1998) and (v) anti-apoptosis engineering which is based on ectopic expression of survival genes (Ishaque and Al-Rubeai, 2002).…”

Section: Discussionmentioning

confidence: 99%

Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

Peng

Fussenegger

2008

Biotech & Bioengineering

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A complex vesicle trafficking system manages the precise and regulated distribution of proteins, membranes and other molecular cargo between cellular compartments as well as the secretion of (heterologous) proteins in mammalian cells. Sec1/Munc18 (SM) proteins are key components of the system by regulating membrane fusion. However, it is not clear how SM proteins contribute to the overall exocytosis. Here, functional analysis of the SM protein Sly1 and Munc18c suggested a united, positive impact upon SNARE-based fusion of ER-to-Golgi- and Golgi-to-plasma membrane-addressed exocytic vesicles and increased the secretory capacity of different therapeutic proteins in Chinese hamster ovary cells up to 40 pg/cell/day. Sly1- and Munc18c-based vesicle traffic engineering cooperated with Xbp-1-mediated ER/Golgi organelle engineering. Our study supports a model for united function of SM proteins in stimulating vesicle trafficking machinery and provides a generic secretion engineering strategy to improve biopharmaceutical manufacturing of important protein therapeutics.

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Section: Discussionmentioning

confidence: 99%

Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

Peng

Fussenegger

2008

Biotech & Bioengineering

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“…RpS6 is a component of the 40S subunit and is the major phosphoprotein of the ribosome, containing five serine residues that are phosphorylated. Ribosomes with the highest degree of RpS6 phosphorylation have an advantage in mobilizing into polysomes, although RpS6 phosphorylation alone may not be sufficient for polysome formation (21,32,53). Phosphorylation of RpS6 tightly correlates with an increase in translation in the cell, particularly of TOP messages (which contain a 5Ј-terminal oligopyrimidine tract).…”

Section: Discussionmentioning

confidence: 99%

Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

Montgomery

Berglund

Beard

et al. 2006

J Virol

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Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.The Alphavirus genus contains over 25 recognized viruses with a wide geographic distribution. Venezuelan equine encephalitis virus (VEE) is a New World alphavirus that is maintained in nature by cycling between a mosquito vector and susceptible vertebrate hosts. VEE is responsible for periodic outbreaks of disease in humans and equines and is classified as a select agent, making it a prominent pathogen among the alphaviruses.Alphaviruses possess a genome of single-stranded messagesense RNA that is approximately 11.5 kb in length. The alphavirus genome is organized such that the 5Ј two-thirds of the genome encodes four nonstructural proteins (nsP1 through nsP4), whereas the 3Ј one-third encodes three mature structural proteins (capsid, E2, and E1) that are expressed at high levels after transcription from an internal 26S subgenomic mRNA promoter. The viral genome appears to be similar to cellular mRNAs, since it contains 5Ј and 3Ј untranslated regions, with a 5Ј-terminal methylguanylate cap and a 3Ј-terminal polyadenylate tail. Upon release into a host cell, the viral genomic RNA is directly translated by the cellular translation machinery. The nonstructural proteins are synthesized as two polyproteins, termed P123 and P1234. P123 results when translation terminates at an opal termination codon between nsP3 and nsP4. When t...

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“…pCS35 and pCS37: In order to enable immunodetection of eIF4G ⌬I -ЈFKBP 3 (pCS8) and eIF4G ⌬II -ЈFKBP 3 (pCS23) they were fused by PCR to a Hemagglutinin A epitope tag (YPYDVPDYA) using oligonucleotides OCS16 (gatcATTAACGCCGGCGCtATTCCA; notI site in bold, beginning of Kozak sequence underlined, annealing sequence in capital letters) or OCS20 (gatcgcggccgCCACCATGAA-CACGCCTTCT; NotI site in bold, Kozak sequence underlined, annealing sequence in capital letters) and OCS17 pSS106, pSS113, and pSS123: Construction of the prokaryotic cloning vectors pSS106, encoding the puromycin resistance conferring gene (pur), pSS113 harboring the SEAP gene as well as the eukaryotic expression vector pSS123 containing the low molecular weight urokinasetype plasminogen activator (u-PA LMW ) have been described before (Schlatter et al, 2002;Schlatter and Fussenegger, 2003).…”

Section: Construction Of Precursor Vectors (Seementioning

confidence: 99%

Modulation of translation‐initiation in CHO‐K1 cells by rapamycin‐induced heterodimerization of engineered eIF4G fusion proteins

Schlatter

Senn

Fussenegger

2003

Biotech & Bioengineering

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Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells.

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Novel CNBP‐ and La‐based translation control systems for mammalian cells

Cited by 31 publications

References 70 publications

Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

Modulation of translation‐initiation in CHO‐K1 cells by rapamycin‐induced heterodimerization of engineered eIF4G fusion proteins

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