AIM: To study the influence of redox environment of Escherichia coli ( E. coli) cytoplasm on disulfide bond formation of recombinant proteins.METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of...AIM: To study the influence of redox environment of Escherichia coli ( E. coli) cytoplasm on disulfide bond formation of recombinant proteins.METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF,and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3)and M15[pREP4]respectively. At the same time, both plasmids were transformedinto a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively.RESULTS: All recombinant E. colistrains could efficiently produce target proteins. The level of BbFGF in BL21(DE3)was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm,and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3)was higher than its counterpart from BL21(DE3). The ED50of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami[pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv]was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from indusion body in M15[pQE-H Fv] was30-35 mg/L and the affinity constant was 1.98×107 mol/L.There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.展开更多
Elastomers with outstanding strength,toughness and healing efficiency are highly promising for many emerging fields.However,it is still a challenge to integrate all these beneficial features in one elastomer.Herein,an...Elastomers with outstanding strength,toughness and healing efficiency are highly promising for many emerging fields.However,it is still a challenge to integrate all these beneficial features in one elastomer.Herein,an asymmetric alicyclic structure adjacent to aromatic disulfide was tactfully introduced into the backbone of polyurethane(PU)elastomer.Specifically,such elastomer(PU-HPS)was fabricated by polycondensing polytetramethylene ether glycol(PTMEG),isophorone diisocyanate(IPDI)and p-hydroxydiphenyl disulfide(HPS)via one-pot method.The molecular mobility and phase morphology of PU-HPS can be tuned by adjusting the HPS content.Consequently,the dynamic exchange of hydrogen and disulfide bonds in the hard segment domains can also be tailored.The optimized sample manifests outstanding tensile strength(46.4 MPa),high toughness(109.1 MJ/m^(3)),high self-healing efficiency after fracture(90.3%),complete scratch recovery(100%)and good puncture resistance.Therefore,this work provides a facile strategy for developing robust self-healing polymers.展开更多
Ubiquitin(Ub) chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub(Ub_3^(11/48)(S-S)), the fir...Ubiquitin(Ub) chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub(Ub_3^(11/48)(S-S)), the first example of an isopeptide mimic for the branched Ub chains,which have recently emerged as an interesting category of Ub modifications. Our strategy comprised the El-dependent synthesis of the Ub conjugate of aminoethanethiol, followed by disulfide formation with Ub(K11 C, K48 C). The structure of the synthetic isopeptide bond mimics was verified by the crystal structure of Ub_3^(11/48)(S-S). Deubiquitination and pulldown assays indicated that the synthetic Ub_3^(11/48)(S-S) could be hydrolyzed by linkage-specific deubiquitinases(K11-specific Cezanne and K48-specific OTUB1), and recognized by proteasomal ubiquitin receptor S5 a.展开更多
Hemophilia A is caused by a genetic mutation in coagulation factor Ⅷ(FⅧ) gene and gene therapy is considered to be a promising strategy for its treatment.We recently demonstrated that co-delivery of two vectors expr...Hemophilia A is caused by a genetic mutation in coagulation factor Ⅷ(FⅧ) gene and gene therapy is considered to be a promising strategy for its treatment.We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor Ⅷ(BDD-FⅧ) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro.In this study,co-injection of both M662C and D1828C mutated BDD-FⅧ gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo.Approximately(239±56) ng mL 1 above endogenous levels of transgenic FⅧ heavy chain was found in mouse plasma using a chain-specific ELISA.For FⅧ coagulation activity,approximately(1.09±0.25) IU mL 1 above endogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay.These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FⅧ gene.These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.展开更多
基金Supported by the National Natural Science Foundation of China,No. 30371661 and No. 30400071and the Natural Science Foundation for Research Team of Guangdong Province, China, No. 2004E039213
文摘AIM: To study the influence of redox environment of Escherichia coli ( E. coli) cytoplasm on disulfide bond formation of recombinant proteins.METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF,and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3)and M15[pREP4]respectively. At the same time, both plasmids were transformedinto a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively.RESULTS: All recombinant E. colistrains could efficiently produce target proteins. The level of BbFGF in BL21(DE3)was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm,and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3)was higher than its counterpart from BL21(DE3). The ED50of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami[pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv]was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from indusion body in M15[pQE-H Fv] was30-35 mg/L and the affinity constant was 1.98×107 mol/L.There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.
基金supported by the National Natural Science Foundation of China(No.51873110)the Foundation of Guangdong Provincial Key Laboratory of Natural Rubber Processing and Key Laboratory of Carb on Fiber and Functio nal Polymers(Beijing University of Chemical Technology),Ministry of Educati on.
文摘Elastomers with outstanding strength,toughness and healing efficiency are highly promising for many emerging fields.However,it is still a challenge to integrate all these beneficial features in one elastomer.Herein,an asymmetric alicyclic structure adjacent to aromatic disulfide was tactfully introduced into the backbone of polyurethane(PU)elastomer.Specifically,such elastomer(PU-HPS)was fabricated by polycondensing polytetramethylene ether glycol(PTMEG),isophorone diisocyanate(IPDI)and p-hydroxydiphenyl disulfide(HPS)via one-pot method.The molecular mobility and phase morphology of PU-HPS can be tuned by adjusting the HPS content.Consequently,the dynamic exchange of hydrogen and disulfide bonds in the hard segment domains can also be tailored.The optimized sample manifests outstanding tensile strength(46.4 MPa),high toughness(109.1 MJ/m^(3)),high self-healing efficiency after fracture(90.3%),complete scratch recovery(100%)and good puncture resistance.Therefore,this work provides a facile strategy for developing robust self-healing polymers.
基金supported by the National Key R&D Program of China(2017YFA0505200)the National Natural Science Foundation of China(91753205,21532004,21761142008)the Program of Introducing Talents of Discipline to Universities of China(B16028)
文摘Ubiquitin(Ub) chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub(Ub_3^(11/48)(S-S)), the first example of an isopeptide mimic for the branched Ub chains,which have recently emerged as an interesting category of Ub modifications. Our strategy comprised the El-dependent synthesis of the Ub conjugate of aminoethanethiol, followed by disulfide formation with Ub(K11 C, K48 C). The structure of the synthetic isopeptide bond mimics was verified by the crystal structure of Ub_3^(11/48)(S-S). Deubiquitination and pulldown assays indicated that the synthetic Ub_3^(11/48)(S-S) could be hydrolyzed by linkage-specific deubiquitinases(K11-specific Cezanne and K48-specific OTUB1), and recognized by proteasomal ubiquitin receptor S5 a.
基金supported by the Natural Science Foundation of Shandong Province,China(Grant No.ZR2010CM061)the Scientific Research Foundation from Ministry of Education for Returned Overseas Chinese Scholars(Grant No.20071108)
文摘Hemophilia A is caused by a genetic mutation in coagulation factor Ⅷ(FⅧ) gene and gene therapy is considered to be a promising strategy for its treatment.We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor Ⅷ(BDD-FⅧ) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro.In this study,co-injection of both M662C and D1828C mutated BDD-FⅧ gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo.Approximately(239±56) ng mL 1 above endogenous levels of transgenic FⅧ heavy chain was found in mouse plasma using a chain-specific ELISA.For FⅧ coagulation activity,approximately(1.09±0.25) IU mL 1 above endogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay.These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FⅧ gene.These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.
