A time- and cost-efficient system for high-level protein production in mammalian cells.
By A. Aricescu, Weixian Lu, E. Jones. Acta Crystallographica Section D: Biological Crystallography, 2006
777 citations recorded by the source corpus.
Most proteins for structural biology studies are produced by high-level expression in Escherichia coli. However, prokaryotic based expression systems fail to generate correctly folded functional forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus-infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N-linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium- or high-throughput pipeline.
Subfields: Biotechnology, Materials Chemistry, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/933b36174184342301be6f09b540c9d784adf993
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Citations in the corpus
- Structure of the decoy module of human glycoprotein 2 and uromodulin and its interaction with bacterial adhesin FimH (2022, methodology): “…levels and FimH L binding experiments, branch region constructs and mutants thereof were expressed in HEK293T cells 20 grown in DMEM medium supplemented with 4 mM l-Gln, 10% FBS and transiently transfected in 4 mM l-Gln, 2% FBS using 25 kDa branched polyethylenimine (Sigma-Aldrich) 19,21 .”
- Structural activation of pro-inflammatory human cytokine IL-23 by cognate IL-23 receptor enables recruitment of the shared receptor IL-12Rβ1 (2017, methodology): “For testing expression of protein constructs and transient expression in HEK293 cells, constructs were cloned into the pHLsec plasmid (Aricescu et al., 2006).”
- Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum (2021, background): “…Cat#CRL-3216 Expi293F Cells GIBCO, Cat#A14527 HEK293T/17 cells ATCC Cat#CRL-11268 HEK293T cells ATCC Cat#CRL-11268 Hamster: ExpiCHO cells Thermo Fisher Cat#A29133 Vero cells ATCC Cat#CCL-81 Recombinant DNA Vector: pHLsec Aricescu et al., 2006 N/A Vector: pNEO Aricescu et al., 2006 N/A Vector: p8.”
- Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion (2016, methodology): “R4V2Q5), was cloned into the pHLSec vector (47).”
- Improving de novo protein binder design with deep learning (2022, neutral): “DNAencodingforthecytokinebindingdomainsofALK(ALK TG-EGFL , residues 648-1030) and LTK (LTK TG-EGFL , residues 63-420) were cloned in the pHLsec vector in frame with a N-terminal chicken RTP μ - like signal peptide sequence and a C-terminal Avi-tag followed by a caspase-3-cleavable Fc-His x6 tag 38 .”