Objective: To obtain recombinant human SDF-1β expressed in E. colt and purify SDF-lfi with bio-logical activity from the bacterium. Methods: A thioredoxin-SDF-1β fusion protein (26×103) composed of230 amino aci...Objective: To obtain recombinant human SDF-1β expressed in E. colt and purify SDF-lfi with bio-logical activity from the bacterium. Methods: A thioredoxin-SDF-1β fusion protein (26×103) composed of230 amino acid residues was expressed in E. coli AD494 (DE3)pLysS under the induction of IPTG whenpET32a( + )-SDF-1β was used as an expression vector. Purified SDF-lfi was produced through following pro-cedures: Bacteria lysis, metal-chelated affinity chromatography (MAC), enterokinase digestion to separateSDF-lfi from fusion protein, cation exchange chromatography (CEC) and reverse-phase high performance liq-uid chromatography (RP-HPLC). Western blot with anti-SDF-1β monoclonal antibody (mAb), N-terminalamino acid sequencing, ligand-binding assay and cytosensor/microphysiometry were used to investigate thebiochemical characters and biological activities of the purified SDF-1β. Results: From 10% to 15% of totalbacterium protein was expressed as fusion protein. Approximately 400 fig purified SDF-1β (7. 8×103) con-sisting of 71 amino acid residues were produced from 1 L of fermented bacteria. Western blot showed that an-ti-SDF-1β mAb bound with the purified SDF-1β specifically. N-terminal amino acid sequencing indicates thatN-terminus of purified SDF-1β possessed as the same amino acid sequence as nature one. Purified SDF-1β notonly had the binding activity with CXCR4 expressing cells [Kd= (12. 20±2. 99) mnol/L], but also activatedCXCR4 expressing cell signaling specifically in a dose-dependence manner. Conclusion: The purified recombi-nant human SDF-1β produced with this method possesses biochemical characters and biological activities assame as those nature human SDF-1β.展开更多
Aflatoxin B_(1)(AFB_(1))is a naturally-occurring mycotoxin and recognized as the most toxic foodborne toxin,particularly causing damages to kidney.Glomerular podocytes are terminally differentiated epithelial cells.AF...Aflatoxin B_(1)(AFB_(1))is a naturally-occurring mycotoxin and recognized as the most toxic foodborne toxin,particularly causing damages to kidney.Glomerular podocytes are terminally differentiated epithelial cells.AFB_(1)induces podocyte inflammation,proteinuria and renal dysfunction.Studying the mechanism of AFB_(1)-induced podocyte inflammation and murine kidney dysfunction,we detected that AFB_(1)increased ubiquitindependent degradation of the transcription factor RelA through enhanced interaction of RelA with E3 ubiquitin ligase tripartite motif containing 7(TRIM7)in mouse podocyte clone-5(MPC-5)and mouse glomeruli.Reduction of RelA resulted in decreasing microRNA-9(miR-9)and activating the chemokine receptor 4(CXCR4),thioredoxin interacting protein(TXNIP),and NOD-like receptor pyrin domain-containing 3(NLRP3)signaling axis(CXCR4/TXNIP/NLRP3 pathway),leading to podocyte inflammation.We also determined that downregulation of miR-9 led to CXCR4 expression and the downstream TXNIP/NLRP3 pathway activation.Overexpression of miR-9 or deletion of CXCR4 suppressed AFB_(1)-induced CXCR4/TXNIP/NLRP3 pathway,resulting in alleviating podocyte inflammation and kidney dysfunction.Our findings indicated that ubiquitin-dependent proteolysis of RelA,downregulation of miR-9,and activation of CXCR4/TXNIP/NLRP3 pathway played an essential role in AFB_(1)-induced glomerular podocyte inflammation.Our study revealed a novel mechanism,via RelA,for the control of AFB_(1)’s nephrotoxicity,leading to an effective protection of food safety and public health.展开更多
文摘Objective: To obtain recombinant human SDF-1β expressed in E. colt and purify SDF-lfi with bio-logical activity from the bacterium. Methods: A thioredoxin-SDF-1β fusion protein (26×103) composed of230 amino acid residues was expressed in E. coli AD494 (DE3)pLysS under the induction of IPTG whenpET32a( + )-SDF-1β was used as an expression vector. Purified SDF-lfi was produced through following pro-cedures: Bacteria lysis, metal-chelated affinity chromatography (MAC), enterokinase digestion to separateSDF-lfi from fusion protein, cation exchange chromatography (CEC) and reverse-phase high performance liq-uid chromatography (RP-HPLC). Western blot with anti-SDF-1β monoclonal antibody (mAb), N-terminalamino acid sequencing, ligand-binding assay and cytosensor/microphysiometry were used to investigate thebiochemical characters and biological activities of the purified SDF-1β. Results: From 10% to 15% of totalbacterium protein was expressed as fusion protein. Approximately 400 fig purified SDF-1β (7. 8×103) con-sisting of 71 amino acid residues were produced from 1 L of fermented bacteria. Western blot showed that an-ti-SDF-1β mAb bound with the purified SDF-1β specifically. N-terminal amino acid sequencing indicates thatN-terminus of purified SDF-1β possessed as the same amino acid sequence as nature one. Purified SDF-1β notonly had the binding activity with CXCR4 expressing cells [Kd= (12. 20±2. 99) mnol/L], but also activatedCXCR4 expressing cell signaling specifically in a dose-dependence manner. Conclusion: The purified recombi-nant human SDF-1β produced with this method possesses biochemical characters and biological activities assame as those nature human SDF-1β.
基金funded by Suzhou Science and Technology Council(SNG201907)Universities Natural Science Foundation of Jiangsu Province(20KJB330002)+6 种基金General Program of China Postdoctoral Science Foundation(2022M711369)the Startup Funding of Soochow University,Jiangsu Province-Suzhou Science and Technology Planning Project(SL T201917)National Natural Science Foundation of China(32172922,31972741)Natural Science Foundation of Jiangsu Province of China(BK20211216,BK20221091)the Startup Funding of Hefei University of Technology(1302003712022058)China-CEEC Joint University Education Project(202010)the Excellence Project PrF UHK(2217/2022-2023)。
文摘Aflatoxin B_(1)(AFB_(1))is a naturally-occurring mycotoxin and recognized as the most toxic foodborne toxin,particularly causing damages to kidney.Glomerular podocytes are terminally differentiated epithelial cells.AFB_(1)induces podocyte inflammation,proteinuria and renal dysfunction.Studying the mechanism of AFB_(1)-induced podocyte inflammation and murine kidney dysfunction,we detected that AFB_(1)increased ubiquitindependent degradation of the transcription factor RelA through enhanced interaction of RelA with E3 ubiquitin ligase tripartite motif containing 7(TRIM7)in mouse podocyte clone-5(MPC-5)and mouse glomeruli.Reduction of RelA resulted in decreasing microRNA-9(miR-9)and activating the chemokine receptor 4(CXCR4),thioredoxin interacting protein(TXNIP),and NOD-like receptor pyrin domain-containing 3(NLRP3)signaling axis(CXCR4/TXNIP/NLRP3 pathway),leading to podocyte inflammation.We also determined that downregulation of miR-9 led to CXCR4 expression and the downstream TXNIP/NLRP3 pathway activation.Overexpression of miR-9 or deletion of CXCR4 suppressed AFB_(1)-induced CXCR4/TXNIP/NLRP3 pathway,resulting in alleviating podocyte inflammation and kidney dysfunction.Our findings indicated that ubiquitin-dependent proteolysis of RelA,downregulation of miR-9,and activation of CXCR4/TXNIP/NLRP3 pathway played an essential role in AFB_(1)-induced glomerular podocyte inflammation.Our study revealed a novel mechanism,via RelA,for the control of AFB_(1)’s nephrotoxicity,leading to an effective protection of food safety and public health.
