Human coagulation Factor V（FV）, together with Factor Xa, assembles to prothrombinase complex on activated cell surface, which converts prothrombin into thrombin, leading to fibrin deposition. The C2 domain of FV is ...Human coagulation Factor V（FV）, together with Factor Xa, assembles to prothrombinase complex on activated cell surface, which converts prothrombin into thrombin, leading to fibrin deposition. The C2 domain of FV is believed to be a primary anchor for the assembly of pro- thrombinase on the cell surface, and was proposed as a target to intervene with pathological thrombotic events. We report here the crystal structure of the C2 domain of FV fused to maltose-binding protein（MBP）. The fusion tag of MBP is critical to generate the crystal for this study. There is no strong interaction between MBP and FVC2. The overall structure of FVC2 is similar to the previous FVC2 structures, suggesting the MBP fusion does not perturb the molecular structure of FVC2. This crystal form of FVC2 can be used for future study of molecular interaction between FVC2 and its inhibitors.展开更多
The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated ...The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated GnRH hexamer gene was inserted into the expression plasmid pMAL-c2x. Recombinant GnRH-6-MBP protein was over- expressed in E.coli strain BL21. Amylose resin with affinity chromatograph was used to purify target protein. The reactiongenicity of fusion protein was identified by indirect enzyme linked immunosorbent assay （ELISA）, and the antigenicity and biological effects of GnRH-6-MBP were tested in mice. In the experiment, 20 male Kunming white mice of 20 d old were randomly divided into treatment and control group. Ten mice were immunized with 100 μg GnRH-6-MBP administered subcutaneously （s.c.） thrice at 2-week intervals with GnRH-6-MBP. Mice were sacrificed after 3 weeks following the booster injection, the testis was removed, weighed and measured, and the histological structure was observed. The reactiongenicity of fusion protein to GnRH antibody was much higher than the control. Active immunization against GnRH-6-MBP reduced remarkably （P 〈 0.01） the length and weight of the testis, and shortened the girth and width of the testis （P 〈 0.05）, and suppressed testicular spermatogenesis compared to the control mice. These results indicate that the recombinant GnRH-6-MBP acted as a strong immunogen and caused atrophy of the testis.展开更多
Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (R...Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (Root-inducing) plasmid was transferred to the plant genome by A. rhizo-genes and has been maintained in all 291 hexaploid sweet potato cultivars of the world. The maintenance in the sweet potato genome and expression of two T-DNA genes for tryptophan-2-monooxygenease (iaaM) and for indole-3-acetamide hydrolase (iaaH) are likely to be physiologically significant since these enzymes convert tryptophan to indole-3-acetic acid, a major plant growth hormone auxin. Sweet potato (Ipomoea batatas (L.) Lam) is ranked the third most important root crop after potato and cassava, and the seventh in global food crop production with more than 126 million metric tons. Although sweet potato originated in Central or South America, China currently produces over 86% of world production with 109 million metric tons. In the United States, North Carolina is the leading producer with 38.5% of the 2007 sweet potato production, followed by California, Mississippi, and Louisiana with 23%, 19%, and 15.9%, respectively. Leaf curl virus diseases have been reported in sweet potato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) belonging to the genus Begomovirus (family Geminiviridae). Although SPLCV does not cause symptoms on Beauregard, one of the most predominant sweet potato cultivars in the US, it can reduce the yield up to 26%. Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E as a fusion protein with maltose-binding protein, and transformed into Escherichia coli strain XL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 μg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltosebinding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the coat protein of SPLCV based on the fact that the mass spectrometry elucidated the sequences corresponding to 37% of amino acid positions of the SPLCV coat protein.展开更多
The bifunctional fusion protein systems consisting of Rhodococcus erythropolis chiral alcohol dehydrogenase(READH),Candida boidinii formate dehydrogenase(CbFDH) or maltose binding protein(MBP) were constructed to rege...The bifunctional fusion protein systems consisting of Rhodococcus erythropolis chiral alcohol dehydrogenase(READH),Candida boidinii formate dehydrogenase(CbFDH) or maltose binding protein(MBP) were constructed to regenerate the cofactors for biocatalysis.READH originated from Rhodococcus erythropolis is an(S)-specific nicotinamide adenine dinucleotide(NADH)-dependent alcohol dehydrogenase,meanwhile,CbFDH originated from Candida boidinii is an NADH-dependent formate dehydrogenase.The strategies of the different fusion protein systems included:(1) fusion of the N terminus of READH to the C terminus of MBP,(2) fusion of the N terminus of CbFDH to the C terminus of MBP,(3) fusion of the N terminus of READH to the C terminus of CbFDH,(4) fusion of the C terminus of READH to the N terminus of CbFDH.The activities of READHs were depressed in all fusion strategies.When the N terminus of READH was fused to the C terminus of CbFDH,READH reached the highest activity,but CbFDH had no activity.In contrast,when the C terminus of READH was fused to the N terminus of CbFDH,CbFDH showed the highest activity,and both moieties displayed activities.From this study,the authors suggest that the rational design of the bifunctional fusion protein system may improve the biocatalysis efficiency by the simultaneous cofactor regeneration.展开更多
基金Supported by the National Natural Science Foundation of China(31161130356)
文摘Human coagulation Factor V（FV）, together with Factor Xa, assembles to prothrombinase complex on activated cell surface, which converts prothrombin into thrombin, leading to fibrin deposition. The C2 domain of FV is believed to be a primary anchor for the assembly of pro- thrombinase on the cell surface, and was proposed as a target to intervene with pathological thrombotic events. We report here the crystal structure of the C2 domain of FV fused to maltose-binding protein（MBP）. The fusion tag of MBP is critical to generate the crystal for this study. There is no strong interaction between MBP and FVC2. The overall structure of FVC2 is similar to the previous FVC2 structures, suggesting the MBP fusion does not perturb the molecular structure of FVC2. This crystal form of FVC2 can be used for future study of molecular interaction between FVC2 and its inhibitors.
基金the support from the Natural Science Foundation of Anhui Provincial Education Department, China (KJ2007B175)
文摘The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated GnRH hexamer gene was inserted into the expression plasmid pMAL-c2x. Recombinant GnRH-6-MBP protein was over- expressed in E.coli strain BL21. Amylose resin with affinity chromatograph was used to purify target protein. The reactiongenicity of fusion protein was identified by indirect enzyme linked immunosorbent assay （ELISA）, and the antigenicity and biological effects of GnRH-6-MBP were tested in mice. In the experiment, 20 male Kunming white mice of 20 d old were randomly divided into treatment and control group. Ten mice were immunized with 100 μg GnRH-6-MBP administered subcutaneously （s.c.） thrice at 2-week intervals with GnRH-6-MBP. Mice were sacrificed after 3 weeks following the booster injection, the testis was removed, weighed and measured, and the histological structure was observed. The reactiongenicity of fusion protein to GnRH antibody was much higher than the control. Active immunization against GnRH-6-MBP reduced remarkably （P 〈 0.01） the length and weight of the testis, and shortened the girth and width of the testis （P 〈 0.05）, and suppressed testicular spermatogenesis compared to the control mice. These results indicate that the recombinant GnRH-6-MBP acted as a strong immunogen and caused atrophy of the testis.
文摘Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (Root-inducing) plasmid was transferred to the plant genome by A. rhizo-genes and has been maintained in all 291 hexaploid sweet potato cultivars of the world. The maintenance in the sweet potato genome and expression of two T-DNA genes for tryptophan-2-monooxygenease (iaaM) and for indole-3-acetamide hydrolase (iaaH) are likely to be physiologically significant since these enzymes convert tryptophan to indole-3-acetic acid, a major plant growth hormone auxin. Sweet potato (Ipomoea batatas (L.) Lam) is ranked the third most important root crop after potato and cassava, and the seventh in global food crop production with more than 126 million metric tons. Although sweet potato originated in Central or South America, China currently produces over 86% of world production with 109 million metric tons. In the United States, North Carolina is the leading producer with 38.5% of the 2007 sweet potato production, followed by California, Mississippi, and Louisiana with 23%, 19%, and 15.9%, respectively. Leaf curl virus diseases have been reported in sweet potato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) belonging to the genus Begomovirus (family Geminiviridae). Although SPLCV does not cause symptoms on Beauregard, one of the most predominant sweet potato cultivars in the US, it can reduce the yield up to 26%. Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E as a fusion protein with maltose-binding protein, and transformed into Escherichia coli strain XL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 μg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltosebinding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the coat protein of SPLCV based on the fact that the mass spectrometry elucidated the sequences corresponding to 37% of amino acid positions of the SPLCV coat protein.
文摘The bifunctional fusion protein systems consisting of Rhodococcus erythropolis chiral alcohol dehydrogenase(READH),Candida boidinii formate dehydrogenase(CbFDH) or maltose binding protein(MBP) were constructed to regenerate the cofactors for biocatalysis.READH originated from Rhodococcus erythropolis is an(S)-specific nicotinamide adenine dinucleotide(NADH)-dependent alcohol dehydrogenase,meanwhile,CbFDH originated from Candida boidinii is an NADH-dependent formate dehydrogenase.The strategies of the different fusion protein systems included:(1) fusion of the N terminus of READH to the C terminus of MBP,(2) fusion of the N terminus of CbFDH to the C terminus of MBP,(3) fusion of the N terminus of READH to the C terminus of CbFDH,(4) fusion of the C terminus of READH to the N terminus of CbFDH.The activities of READHs were depressed in all fusion strategies.When the N terminus of READH was fused to the C terminus of CbFDH,READH reached the highest activity,but CbFDH had no activity.In contrast,when the C terminus of READH was fused to the N terminus of CbFDH,CbFDH showed the highest activity,and both moieties displayed activities.From this study,the authors suggest that the rational design of the bifunctional fusion protein system may improve the biocatalysis efficiency by the simultaneous cofactor regeneration.
