Transient Expression in HEK 293 Cells: An Alternative to E. coli for the Production of Secreted and Intracellular Mammalian Proteins

Nettleship, Joanne E.; Watson, Peter J.; Rahman-Huq, Nahid; Fairall, Louise; Posner, Mareike G.; Upadhyay, Abhishek; Reddivari, Yamini; Chamberlain, Jonathan M. G.; Kolstoe, Simon; Bagby, Stefan; Schwabe, John W. R.; Owens, Raymond J.

doi:10.1007/978-1-4939-2205-5_11

Cited by 43 publications

(31 citation statements)

References 29 publications

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“…After 18-20 h, 150 lL of ExpiFectamine TM transfection Enhancer 1 and 1.5 mL of ExpiFectamine Transfection TM Enhancer 2 were added to the transfected cells. The supernatant containing the mENPP2-1-8xHis protein was harvested after 96 h. Secreted protein was purified by automated immobilized metal affinity chromatography followed by gel filtration chromatography using the method of Nettleship et al [60,61]. Briefly, 200 mL of sample was loaded onto a 5 mL HisTrap FF column (GE Healthcare) before washing with 50 mL of 50 mM Tris, pH 7.5, 500 mM NaCl, 30 mM imidazole.…”

Section: Methodsmentioning

confidence: 99%

ENPP1 processes protein ADP‐ribosylation in vitro

et al. 2016

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ADP‐ribosylation is a conserved post‐translational protein modification that plays a role in all major cellular processes, particularly DNA repair, transcription, translation, stress response and cell death. Hence, dysregulation of ADP‐ribosylation is linked to the physiopathology of several human diseases including cancers, diabetes and neurodegenerative disorders. Protein ADP‐ribosylation can be reversed by the macrodomain‐containing proteins PARG, TARG1, MacroD1 and MacroD2, which hydrolyse the ester bond known to link proteins to ADP‐ribose as well as consecutive ADP‐ribose subunits; targeting this bond can thus result in the complete removal of the protein modification or the conversion of poly(ADP‐ribose) to mono(ADP‐ribose). Recently, proteins containing the NUDIX domain – namely human NUDT16 and bacterial RppH – have been shown to process in vitro protein ADP‐ribosylation through an alternative mechanism, converting it into protein‐conjugated ribose‐5′‐phosphate (R5P, also known as pR). Though this protein modification was recently identified in mammalian tissues, its physiological relevance and the mechanism of generating protein phosphoribosylation are currently unknown. Here, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as the first known mammalian enzyme lacking a NUDIX domain to generate pR from ADP‐ribose on modified proteins in vitro. Thus, our data show that at least two enzyme families – Nudix and ENPP/NPP – are able to metabolize protein‐conjugated ADP‐ribose to pR in vitro, suggesting that pR exists and may be conserved from bacteria to mammals. We also demonstrate the utility of ENPP1 for converting protein‐conjugated mono(ADP‐ribose) and poly(ADP‐ribose) into mass spectrometry‐friendly pR tags, thus facilitating the identification of ADP‐ribosylation sites.

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Section: Methodsmentioning

confidence: 99%

ENPP1 processes protein ADP‐ribosylation in vitro

et al. 2016

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“…GTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCCC-3'. The genes were inserted into the pOPIN expression vectors by Infusion® cloning.The CR3022 hIgG1 heavy chain gene was amplified through joining three fragments (using the forward primer 5'-GCGTAGCTGAAACCGGCCAGATGCAGCTGGTGCAATC-3vector pOPINTTGneo(Nettleship et al, 2015) incorporating a C-terminal His6 tag.. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a 1 4CR3022 used for neutralisation: The heavy and kappa light variable genes of the antibody were sourced from the Genbank ABA54613.1 and ABA54614.1 respectively and the codon optimised sequences were synthesized by GeneArt.…”

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confidence: 99%

Neutralization of SARS-CoV-2 by destruction of the prefusion Spike

Huo

Zhao

Ren

et al. 2020

Preprint

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Highlights • CR3022 neutralises SARS-CoV-2 • Neutralisation is by destroying the prefusion SPIKE conformation • This antibody may have therapeutic potential alone or with one blocking receptor attachment Summary There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.

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“…Transient expression platforms for the production of mammalian proteins often use human HEK-293 or hamster CHO cells [25,26]. HEK-293 is a cell line derived from human embryonic kidney cells grown in tissue culture and is widely used in cell biology and biotechnology [27].…”

Section: Transient Expression Systems For Recombinant Proteinsmentioning

confidence: 99%

Over-Production of Therapeutic Growth Factors for Cartilage Regeneration by Protein Production Platforms and Protein Packaging Cell Lines: A Narrative Review of the Current State-of-the-Art

Mobasheri¹,

Martín‐Vasallo²

2019

Preprint

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This article focuses on the current state-of-the-art in the area of cellular and molecular biotechnology for over-production of clinically relevant therapeutic growth factors and how the technology can be used for the treatment of osteoarthritis (OA). Transfected and irradiated protein packaging cell lines may be used as “cellular factories” for large-scale production of therapeutic proteins and pro-anabolic growth factors, particularly in the context of cartilage matrix regeneration. We discuss the potential for new innovations in regenerative medicine for degenerative diseases of synovial joints using mammalian protein production platforms, specifically protein packaging cell lines, for over-producing growth factors for cartilage tissue regeneration and give recent examples. Mammalian protein production platforms that incorporate protein packaging cell lines are superior to bacterial expression systems and are likely to have a significant impact on the development of new biological therapies for treating focal cartilage defects and more generally for the treatment of degenerative joint diseases such as OA.

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Transient Expression in HEK 293 Cells: An Alternative to E. coli for the Production of Secreted and Intracellular Mammalian Proteins

Cited by 43 publications

References 29 publications

ENPP1 processes protein ADP‐ribosylation in vitro

ENPP1 processes protein ADP‐ribosylation in vitro

Neutralization of SARS-CoV-2 by destruction of the prefusion Spike

Over-Production of Therapeutic Growth Factors for Cartilage Regeneration by Protein Production Platforms and Protein Packaging Cell Lines: A Narrative Review of the Current State-of-the-Art

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