Producing GST-Cbx7 Fusion Proteins from Escherichia coli

Huynh, Thao Ngoc; Ren, Xiaojun

doi:10.21769/bioprotoc.2333

Cited by 4 publications

(3 citation statements)

References 9 publications

(15 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Protein concentration was determined using a bovine serum albumin (BSA) protein assay kit (Kang Wei, China). The specificity of sCD83 protein was analyzed by sandwich ELISA (62).…”

Section: Methodsmentioning

confidence: 99%

Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83

Chen

Bai

Liu

et al. 2018

J Virol

View full text Add to dashboard Cite

Porcine reproductive and respiratory syndrome virus (PRRSV), a virulent pathogen of swine, suppresses the innate immune response and induces persistent infection. One mechanism used by viruses to evade the immune system is to cripple the antigen-processing machinery in monocyte-derived dendritic cells (MoDCs). In this study, we show that MoDCs infected by PRRSV express lower levels of the major histocompatibility complex (MHC)-peptide complex proteins TAP1 and ERp57 and are impaired in their ability to stimulate T cell proliferation and increase their production of CD83. Neutralization of sCD83 removes the inhibitory effects of PRRSV on MoDCs. When MoDCs are incubated with exogenously added sCD83 protein, TAP1 and ERp57 expression decreases and T lymphocyte activation is impaired. PRRSV nonstructural protein 1α (Nsp1α) enhances CD83 promoter activity. Mutations in the ZF domain of Nsp1α abolish its ability to activate the CD83 promoter. We generated recombinant PRRSVs with mutations in Nsp1α and the corresponding repaired PRRSVs. Viruses with Nsp1α mutations did not decrease levels of TAP1 and ERp57, impair the ability of MoDCs to stimulate T cell proliferation, or increase levels of sCD83. We show that the ZF domain of Nsp1α stimulates the secretion of CD83, which in turn inhibits MoDC function. Our study provides new insights into the mechanisms of immune suppression by PRRSV. PRRSV has a severe impact on the swine industry throughout the world. Understanding the mechanisms by which PRRSV infection suppresses the immune system is essential for a robust and sustainable swine industry. Here, we demonstrated that PRRSV infection manipulates MoDCs by interfering with their ability to produce proteins in the MHC-peptide complex. The virus also impairs the ability of MoDCs to stimulate cell proliferation, due in large part to the enhanced release of soluble CD83 from PRRSV-infected MoDCs. The viral nonstructural protein 1 (Nsp1) is responsible for upregulating CD83 promoter activity. Amino acids in the ZF domain of Nsp1α (L5-2A, rG45A, G48A, and L61-6A) are essential for CD83 promoter activation. Viruses with mutations at these sites no longer inhibit MoDC-mediated T cell proliferation. These findings provide novel insights into the mechanism by which the adaptive immune response is suppressed during PRRSV infection.

show abstract

“…Protein concentration was determined using a bovine serum albumin (BSA) protein assay kit (Kang Wei, China). The specificity of sCD83 protein was analyzed by sandwich ELISA (62).…”

Section: Methodsmentioning

confidence: 99%

Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83

Chen

Bai

Liu

et al. 2018

J Virol

View full text Add to dashboard Cite

show abstract

“…Producing GST-Cbx7 fusion proteins is described in more detail at Bio-protocol ( Huynh and Ren, 2017 ). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells.…”

Section: Methodsmentioning

confidence: 99%

Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

Zhen

Tatavosian

Huynh

et al. 2016

eLife

Self Cite

100

107

View full text Add to dashboard Cite

The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.DOI: http://dx.doi.org/10.7554/eLife.17667.001

show abstract

“…Recombinant CBX2 and its variants were generated and purified according to previous reports with modifications (81). The pGEX-6P-1-GST-FLAG vector containing the Cbx2 fusion gene was transformed into Rosetta TM 2 (pLysS) host strains (71403, Novagen).…”

Section: Generating Recombinant Protein Of Cbx2 and Its Variantsmentioning

confidence: 99%

Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

Tatavosian

Kent

Brown

et al. 2019

Journal of Biological Chemistry

Self Cite

272

280

View full text Add to dashboard Cite

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

show abstract

Producing GST-Cbx7 Fusion Proteins from Escherichia coli

Cited by 4 publications

References 9 publications

Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83

Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83

Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

Contact Info

Product

Resources

About