To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a pro...To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.展开更多
AIM:To investigate whether human acyl-CoA synthetase 5(ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS:The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expre...AIM:To investigate whether human acyl-CoA synthetase 5(ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS:The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes.Ni 2+-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C.In addition,ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored.ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence.The ACSL assay mix included TrisHCl(pH 7.4),ATP,CoA,EDTA,DTT,MgCl 2,[9,103 H] palmitic acid,and triton X-100.The 200 μL reaction was initiated with the addition of solubilized,purified recombinant proteins or cellular lysates.Reactions were terminated after 10,30 or 60 min of incubation with Doles medium.RESULTS:Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl betaD-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni 2+-affinity chromatography.Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5,as well as recombinant rat ACSL1(sensitive control),but not recombinant rat ACSL5(insensitive control).The IC50 for human ACSL5 was about 10 μmol/L.The inhibitory triacsin C effect was similar for different incubation times(10,30 and 60 min) and was not modified by the N-or C-terminal location of the 6xHis-tag.In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment,stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed.In both models,ACSL5 peak activity was found at pH 7.5 and pH 9.5,corresponding to the properties of recombinant human ACSL5 protein.In the presence of triacsin C(25 μmol/L),total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.CONCLUSION:The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.展开更多
The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin prote...The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.展开更多
基金The National Basic Research Program of China(973Program)(No.2011CB933503)the National Natural Science Foundation of China for Key Project of International Cooperation(No.61420106012)China Postdoctoral Science Foundation(No.2013M541592)
文摘To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.
基金Supported by Deutsche Forschungsgemeinschaft, No. GA785/6-1Deutsche Krebshilfe, No. 109313the Rotationsprogramm of the Medical Faculty RWTH Aachen University (to Kaemmerer E)
文摘AIM:To investigate whether human acyl-CoA synthetase 5(ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS:The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes.Ni 2+-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C.In addition,ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored.ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence.The ACSL assay mix included TrisHCl(pH 7.4),ATP,CoA,EDTA,DTT,MgCl 2,[9,103 H] palmitic acid,and triton X-100.The 200 μL reaction was initiated with the addition of solubilized,purified recombinant proteins or cellular lysates.Reactions were terminated after 10,30 or 60 min of incubation with Doles medium.RESULTS:Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl betaD-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni 2+-affinity chromatography.Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5,as well as recombinant rat ACSL1(sensitive control),but not recombinant rat ACSL5(insensitive control).The IC50 for human ACSL5 was about 10 μmol/L.The inhibitory triacsin C effect was similar for different incubation times(10,30 and 60 min) and was not modified by the N-or C-terminal location of the 6xHis-tag.In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment,stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed.In both models,ACSL5 peak activity was found at pH 7.5 and pH 9.5,corresponding to the properties of recombinant human ACSL5 protein.In the presence of triacsin C(25 μmol/L),total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.CONCLUSION:The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.
基金Supported by the Dalian Municipal Government of China (No. 2007B11NC069)the Key Laboratory Foundation of the Educational Department of Liaoning Province (No.2009S024)the Grant of Dalian Fisheries University (No. SY2007005)
文摘The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene （tTaA） from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can he used for further functional and structural studies.
