To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C termin...To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.展开更多
The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative...The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus （rt306P） and the polymerase gene from a mutant, with rt306P substituted by serine （rtP306S） were separately fused to the maltose binding protein （MBP） gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Westem blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.展开更多
A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previou...A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysregulated in neuropsychiatric disorders and cognitive dysfunction:apolipoprotein H(APOH),apolipoprotein J(APOJ),apolipoprotein A4(APOA4),apolipoprotein E(APOE),and apolipoprotein D(APOD).The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOFTM 5600,followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500.Our results showed that the collision energies of mTRAQ-labeled peptides were approximately 15%–20%higher than corresponding non-labeled peptides.Through optimized transitions and parameters,we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins.The results indicated that the MRM signals of four target APOs were significantly increased after depletion,while the MRM signal of one APO,APOD,was decreased.Furthermore,the relative abundances of the five target APOs in healthy human plasma were stable,and the ranking of these proteins according to their MS responses changed slightly.Therefore,we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma.展开更多
In the research of bio-molecular chips and sensors, extra electric biases are most often employed to control and manipulate the DNA and protein molecules moving through micro/nano-fluidic channels. In order to accurat...In the research of bio-molecular chips and sensors, extra electric biases are most often employed to control and manipulate the DNA and protein molecules moving through micro/nano-fluidic channels. In order to accurately and flexibly control the bio-molecules as they move within the channels, a clear understanding of how the current changes within the buffer solution caused by an applied bias is fundamental. In this report, the current changed value of different buffer solutions, e.g., KC1, TE, and TBE was systematically studied with real-time monitoring and quantitative analysis in the situation of the buffers moving through a fluidic channel with a 5 μm inner diameter, driven by biases of 50 or 100 mV. The results revealed that the relation- ship between the current changed value and the pause interval of the applied electric field is highly consistent with the Hill Equation, which is helpful for accurately detecting and manipulating single biomolecules in microfluidic sensors and biochips.展开更多
In this work,we parallelly detected the specific binding between microarray targets including 12 different kinds of proteins and the probe solution containing five corresponding antibodies and quantitatively analyzed ...In this work,we parallelly detected the specific binding between microarray targets including 12 different kinds of proteins and the probe solution containing five corresponding antibodies and quantitatively analyzed the interactions between CDH13 and solution phase anti-CDH13 at six different probe concentrations by oblique-incidence reflectivity difference(OIRD)method in label-free format.The detection sensitivity reached 10 ng/mL.The experimental results indicate that the OIRD method is a promising and competing technique not only in research work but also in clinic.展开更多
Papillon-Lefevre Syndrome is a rare autosomal recessive disorder characterized by rapidly progressive periodontitis and confined palrnoplantar hyperkeratosis resulting from genetic mutations in cathepsin C （CTSC）. T...Papillon-Lefevre Syndrome is a rare autosomal recessive disorder characterized by rapidly progressive periodontitis and confined palrnoplantar hyperkeratosis resulting from genetic mutations in cathepsin C （CTSC）. The present study investigated the effect of CTSC on keratinocyte proliferation and apoptosis. HaCaT keratinocytes were transfected with wild-type CTSC and CTSC-targeted siRNAs to investigate the effects of CTSC expression on cell keratosis. Real-time PCR and Western blot analyses showed that the levels of loricrin and keratin （KRT）-I, but not KRT9, was correlated with CTSC expression. Loricrin was increased in the CTSC-overex- pression group and downregulated in the CTSC-silenced group. A positive association between loricrin expression and cell apoptosis was detected in HaCaT keratinocytes. KRT1 was decreased in the CTSC-overexpression group and increased in the CTSC-silenced group. Prominent, punctuate KRT1 aggregates were present in CTSC-knockdown HaCaT cells. This study suggested that loss of CTSC contributes to keratinocyte hyperkeratosis via downregulation of loricrin and enhanced cell proliferation.展开更多
基金Supported by the National Natural Science Foundation of China (No.20336010 and No.20176025).
文摘To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.
基金grants from China National 973 Project （Grant G 1999054105） National Natural Science Foundation of China （No. 30530040）.
文摘The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus （rt306P） and the polymerase gene from a mutant, with rt306P substituted by serine （rtP306S） were separately fused to the maltose binding protein （MBP） gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Westem blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.
基金supported by the National Basic Research Program of China(2009CB918300)the National Natural Science Foundation of China(31271189 and 81101009)
文摘A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysregulated in neuropsychiatric disorders and cognitive dysfunction:apolipoprotein H(APOH),apolipoprotein J(APOJ),apolipoprotein A4(APOA4),apolipoprotein E(APOE),and apolipoprotein D(APOD).The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOFTM 5600,followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500.Our results showed that the collision energies of mTRAQ-labeled peptides were approximately 15%–20%higher than corresponding non-labeled peptides.Through optimized transitions and parameters,we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins.The results indicated that the MRM signals of four target APOs were significantly increased after depletion,while the MRM signal of one APO,APOD,was decreased.Furthermore,the relative abundances of the five target APOs in healthy human plasma were stable,and the ranking of these proteins according to their MS responses changed slightly.Therefore,we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma.
基金supported by the Major Research Plan of the National Natural Science Foundation of China(Grant No.91123030)the Interna-tional Cooperation Foundation of the National Science and Technology Major Project of the Ministry of Science and Technology of China(Grant No.2011DFA12220)the National Natural Science Foundation of China(Grant No.61378083)
文摘In the research of bio-molecular chips and sensors, extra electric biases are most often employed to control and manipulate the DNA and protein molecules moving through micro/nano-fluidic channels. In order to accurately and flexibly control the bio-molecules as they move within the channels, a clear understanding of how the current changes within the buffer solution caused by an applied bias is fundamental. In this report, the current changed value of different buffer solutions, e.g., KC1, TE, and TBE was systematically studied with real-time monitoring and quantitative analysis in the situation of the buffers moving through a fluidic channel with a 5 μm inner diameter, driven by biases of 50 or 100 mV. The results revealed that the relation- ship between the current changed value and the pause interval of the applied electric field is highly consistent with the Hill Equation, which is helpful for accurately detecting and manipulating single biomolecules in microfluidic sensors and biochips.
基金supported by the National Basic Research Program of China(Grant No.2007CB935700)
文摘In this work,we parallelly detected the specific binding between microarray targets including 12 different kinds of proteins and the probe solution containing five corresponding antibodies and quantitatively analyzed the interactions between CDH13 and solution phase anti-CDH13 at six different probe concentrations by oblique-incidence reflectivity difference(OIRD)method in label-free format.The detection sensitivity reached 10 ng/mL.The experimental results indicate that the OIRD method is a promising and competing technique not only in research work but also in clinic.
文摘Papillon-Lefevre Syndrome is a rare autosomal recessive disorder characterized by rapidly progressive periodontitis and confined palrnoplantar hyperkeratosis resulting from genetic mutations in cathepsin C （CTSC）. The present study investigated the effect of CTSC on keratinocyte proliferation and apoptosis. HaCaT keratinocytes were transfected with wild-type CTSC and CTSC-targeted siRNAs to investigate the effects of CTSC expression on cell keratosis. Real-time PCR and Western blot analyses showed that the levels of loricrin and keratin （KRT）-I, but not KRT9, was correlated with CTSC expression. Loricrin was increased in the CTSC-overex- pression group and downregulated in the CTSC-silenced group. A positive association between loricrin expression and cell apoptosis was detected in HaCaT keratinocytes. KRT1 was decreased in the CTSC-overexpression group and increased in the CTSC-silenced group. Prominent, punctuate KRT1 aggregates were present in CTSC-knockdown HaCaT cells. This study suggested that loss of CTSC contributes to keratinocyte hyperkeratosis via downregulation of loricrin and enhanced cell proliferation.
