Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI

Tumuluri, Vinayak Sadasivam; Saikrishnan, K.

doi:10.21769/bioprotoc.4275

Cited by 2 publications

(3 citation statements)

References 7 publications

(6 reference statements)

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“…The plasmid pET‐30a‐rHSP40 was transfected into Escherichia coli BL21 (DE3) to express rHSP40. rHSP40 was purified using a Ni‐NTA Purification System (ThermoFisher Scientific) 30 . Residual LPS was detected using an endotoxin detection kit (CAT# EC80545, BIOENDO) according to the manufacture's instruction.…”

Section: Methodsmentioning

confidence: 99%

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Heat shock protein 40 of Streptococcus pneumoniae induces immune response of human dendritic cells via TLR4‐dependent p38 MAPK and JNK signaling pathways

Liu

Lin

Chen

2022

Immunity Inflam & Disease

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Introduction: Heat shock protein 40 (HSP40) is a vaccine adjuvant candidate for Streptococcus pneumoniae. The mechanism by which HSP40 activates the human dendritic cells (DCs) is unclear.Methods: DCs were isolated from human peripheral blood and their markers (HLA-DR, CD86, CD83, and CD80) were detected by flow cytometry. The messenger RNA (mRNA) and secretion levels of inflammary cytokines were measured after DCs were stimulated with recombinant HSP40 (rHSP40). Short hairpin RNAs were used to knock down toll-like receptor 2 (TLR2) and TLR4. The TLR2-or TLR4-deficient DCs were treated with lipopolysaccharides, rHSP40, or peptidoglycan, and then the secretion levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured. Moreover, the secretion levels of TNF-α and IL-6 were measured after DCs were treated with mitogen-activated protein kinase (MAPK) inhibitors including SB203580, SP600125, and U0126. In addition, the phosphorylation levels of p38 MAPK and Jun N-terminal kinase (JNK) in DC cells were determined using western blot analysis after treatment with rHSP40 for different times.Results: DCs were successfully isolated and cultured. rHSP40 treatment significantly increased cytokine levels in a concentration-dependent manner. TLR4 deficiency, but not TLR2 deficiency, significantly suppressed the rHSP40-induced secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). SB203580 and SP600125 significantly inhibited the rHSP40-induced secretion of TNF-α and IL-6. rHSP40 significantly enhanced the phosphorylation of p38 MAPK and JNK.Conclusion: HPS40 stimulates the immune response of DCs via the p38 MAPK and JNK signaling pathways, which depend on TLR4.

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Section: Methodsmentioning

confidence: 99%

“…rHSP40 was purified using a Ni‐NTA Purification System (ThermoFisher Scientific). 30 Residual LPS was detected using an endotoxin detection kit (CAT# EC80545, BIOENDO) according to the manufacture's instruction. The concentration of rHSP40 was detected using a Detergent Compatible Bradford Protein Assay kit (CAT# P0006C, Beyotime).…”

Section: Methodsmentioning

confidence: 99%

Heat shock protein 40 of Streptococcus pneumoniae induces immune response of human dendritic cells via TLR4‐dependent p38 MAPK and JNK signaling pathways

Liu

Lin

Chen

2022

Immunity Inflam & Disease

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“…SauUSI and the nuclease-inactive mutant (SauUSI H119A ) were purified using the protocol described earlier (17, 24). Both the sauUSI and sauUSI H119A were cloned into a pHis17 expressed vector containing a C-terminal 6x His-Tag.…”

Section: Methodsmentioning

confidence: 99%

ATP-dependent restriction enzyme SauUSI fromStaphylococcus aureusis also abona fidesingle strand DNA endonuclease

Tumuluri

Saikrishnan

2022

Preprint

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Restriction endonucleases cleave exogenous DNA thus restricting horizontal gene transfer and phage infection of host bacterium. This nucleolytic activity occurs on double-stranded DNA (dsDNA) and is target site specific. Here we report that the Type IV ATP-dependent restriction endonuclease SauUSI from Staphylococcus aureus also possesses a hitherto unknown single-stranded DNA (ssDNA) endonuclease activity. We demonstrate that, unlike the dsDNA cleavage activity, ssDNA cleavage by SauUSI does not require divalent cation or ATP hydrolysis and is target-site and DNA methylation-status independent. Furthermore, we show that SauUSI can cut ssDNA gaps, overhangs, bubbles and loops but not ssRNA. The activity is inhibited at higher concentrations of magnesium ion, ATP, and the presence of single strand DNA binding protein. The ssDNA nuclease activity is thus tightly regulated and may protect the host DNA from damage by SauUSI.

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Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI

Cited by 2 publications

References 7 publications

Heat shock protein 40 of Streptococcus pneumoniae induces immune response of human dendritic cells via TLR4‐dependent p38 MAPK and JNK signaling pathways

Heat shock protein 40 of Streptococcus pneumoniae induces immune response of human dendritic cells via TLR4‐dependent p38 MAPK and JNK signaling pathways

ATP-dependent restriction enzyme SauUSI fromStaphylococcus aureusis also abona fidesingle strand DNA endonuclease

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