Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Portolano, Nicola; Watson, Peter J.; Fairall, Louise; Millard, Christopher; Milano, Charles P.; Song, Yun; Cowley, Shaun M.; Schwabe, John W. R.

doi:10.3791/51897

Cited by 55 publications

(56 citation statements)

References 12 publications

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“…Compared with the glycosylation patterns obtained using bac-to-bac or other expression systems, that of proteins expressed by a mammalian cell line represents a more physiological approach to study CD97 adhesion [42] . Although the EGF1-4 fragment can be expressed at the same level as WT CD97ECD, the reduced expression of EGF1-5 and lack of secretion of the GAIN domain indicate that the EGF5 and GAIN domains may directly interact with each other.…”

Section: Discussionmentioning

confidence: 99%

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

et al. 2016

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CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAINtruncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from ...

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Section: Discussionmentioning

confidence: 99%

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

et al. 2016

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“…The development of human cell lines that grow well in suspension (e.g. HEK293F) and synthetic media that compares favorably to the cost of traditional FBS‐supplemented growth media have made large scale growth of mammalian cells feasible . Transient expression of recombinant proteins by transfecting plasmid DNA can be used to express recombinant proteins before toxic effects overwhelm a culture.…”

Section: Introductionmentioning

confidence: 99%

Selectable high‐yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

et al. 2018

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Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.

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“…Due to their clear advantage in terms of yield and cost, simple prokaryotic expression systems, particularly Escherichia coli , or lower eukaryotic hosts, such as yeast, have long dominated the field of recombinant protein expression [17]. However, mammalian expression systems that are capable of providing native protein folding, and natural posttranslational modifications, have been increasingly used in the past years to reliably express proteins in their native conformation [18]. …”

Section: Introductionmentioning

confidence: 99%

Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization

et al. 2016

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BackgroundDue to their rising incidence and progressive geographical spread, infections with mosquito-borne viruses, such as dengue (DENV), chikungunya and zika virus, have developed into major public health challenges. Since all of these viruses may cause similar symptoms and can occur in concurrent epidemics, tools for their differential diagnosis and epidemiological monitoring are of urgent need.ResultsHere we report the application of a novel strategy to rapidly generate monoclonal antibodies (mAbs) against native viral antigens, exemplified for the DENV nonstructural glycoprotein 1 (NS1). The described system is based on the immunization of mice with transfected mammalian cells expressing the target antigens in multiple displays on their cell surface and thereby presenting them efficiently to the host immune system in their native conformation. By applying this cell-based approach to the DENV NS1 protein of serotypes 1 (D1NS1) and 4 (D4NS1), we were able to rapidly generate panels of DENV NS1 serotype cross-reactive, as well as D1NS1- and D4NS1 serotype-specific mAbs. Our data show that the generated mAbs were capable of recognizing the endogenous NS1 protein in DENV-containing biological samples.ConclusionThe use of this novel immunization strategy, allows for a fast and efficient generation of hybridoma cell lines, producing mAbs against native viral antigens. Envisaged applications of the mAbs include the development of test platforms enabling a differentiation of the DENV serotypes and high resolution immunotyping for epidemiological studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0314-5) contains supplementary material, which is available to authorized users.

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Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Cited by 55 publications

References 12 publications

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

Selectable high‐yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization

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