By using the recombinant DNA technology, the gene of the bovine mature prion protein bPrPcL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L ...By using the recombinant DNA technology, the gene of the bovine mature prion protein bPrPcL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L urea, the expression product was purified by cation ion exchange chromatography. The purified product was refolded by dilution and the recovery was about 15%. Analysis of mass spectrum, circular dichroism (CD) spectrum and Fourier transform infrared (FTIR) spectrum demonstrate that the molecular weight of the bPrPcL is 23 630 u, the bPrPcL has a high α-helix content (36.1%) and low (3-sheet content (11.9%).展开更多
An orthorhombic crystal form of a recombi-nant yeast prion protein with shortened N-terminal, 90Ure2p, has been obtained. Crystals were grown by the vapordiffu-sion technique against a mother liquor containing imidazo...An orthorhombic crystal form of a recombi-nant yeast prion protein with shortened N-terminal, 90Ure2p, has been obtained. Crystals were grown by the vapordiffu-sion technique against a mother liquor containing imidazole. Crystals belong to the primitive orthorhombic lattice with the cell parameters a = 54.5 A, b = 74.7 A, c = 131.0 A. The crystals diffract to beyond 3.0 A resolution at a synchrotron beamline.展开更多
African swine fever virus(ASFV)is a giant and complex DNA virus that causes a highly contagious and lethal swine disease for which there is no vaccine available.Here we describe the cryo—electron microscopy(cryo-EM)s...African swine fever virus(ASFV)is a giant and complex DNA virus that causes a highly contagious and lethal swine disease for which there is no vaccine available.Here we describe the cryo—electron microscopy(cryo-EM)structure of ASFV,the first virus(to our knowledge)that has been found to use two lipid membrane layers and two protein shells to encapsidate and protect its genome.Using an optimized image reconstruction strategy,we solved the AFSV capsid structure up to 4.1-angstroms,which is built from 17,280 proteins,including one major(MCP)and four minor capsid proteins(M1249 L,p17,p49 and H240 R),and organized into pentasymmetrons and trisymmetrons.The atomic structure of the MCP informs putative conformational epitopes,which determine the specific differences between the virus types,being valuable for epitope-focused immunogen design against ASFV infection.The minor capsid proteins form a complicated network below the outer capsid shell,stabilizing the capsid by holding adjacent capsomers together.Acting as core organizers,100-nm-length M1249 L proteins,running along each edge of the trisymmetrons and bridging two neighboring pentasymmtrons,form extensive intermolecular networks with other capsid proteins to guide the formation of capsid framework.These structural details unveil the basis of capsid stability and assembly,opening up new avenues for ASF vaccine development.展开更多
In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se-Met) derivative of TTR was overexpressed in E. coli stra...In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se-Met) derivative of TTR was overexpressed in E. coli strain M15, with pQE-30 plasmid, through IPTG induction in M9 medium containing Se-Met. The product was purified to an estimated homogeneity of greater than 95% according to SDS-PAGE, by a Ni-NTA metal affinity followed by a Mono Q anion exchange column chromatography. The successful substitution of Se-Met for methionine (Met) was confirmed by MALDI-TOF and ESI-Quadrupole Mass Spectrometry analysis. The derivative crystal was obtained using similar conditions as those for the native.展开更多
文摘By using the recombinant DNA technology, the gene of the bovine mature prion protein bPrPcL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L urea, the expression product was purified by cation ion exchange chromatography. The purified product was refolded by dilution and the recovery was about 15%. Analysis of mass spectrum, circular dichroism (CD) spectrum and Fourier transform infrared (FTIR) spectrum demonstrate that the molecular weight of the bPrPcL is 23 630 u, the bPrPcL has a high α-helix content (36.1%) and low (3-sheet content (11.9%).
基金This work was supported by the National NaturalScience Foundation of China (Grant Nos. 39870174 and 39970155) the State "863" High-Tech Project (Grant No. 103130306) and "973" Project (Grant Nos. G1999075602, G1999011902 and 1998051105).
文摘An orthorhombic crystal form of a recombi-nant yeast prion protein with shortened N-terminal, 90Ure2p, has been obtained. Crystals were grown by the vapordiffu-sion technique against a mother liquor containing imidazole. Crystals belong to the primitive orthorhombic lattice with the cell parameters a = 54.5 A, b = 74.7 A, c = 131.0 A. The crystals diffract to beyond 3.0 A resolution at a synchrotron beamline.
基金supported by the Strategic Priority Research Program(XDB08020200)the Key Programs of the Chinese Academy of Science(KJZD-SW-L05)+2 种基金the National Key Research and Development Program(2017YFC0840300)the National Natural Science Foundation of China(31800145and 31570717)supported by Ten Thousand Talent Program。
文摘African swine fever virus(ASFV)is a giant and complex DNA virus that causes a highly contagious and lethal swine disease for which there is no vaccine available.Here we describe the cryo—electron microscopy(cryo-EM)structure of ASFV,the first virus(to our knowledge)that has been found to use two lipid membrane layers and two protein shells to encapsidate and protect its genome.Using an optimized image reconstruction strategy,we solved the AFSV capsid structure up to 4.1-angstroms,which is built from 17,280 proteins,including one major(MCP)and four minor capsid proteins(M1249 L,p17,p49 and H240 R),and organized into pentasymmetrons and trisymmetrons.The atomic structure of the MCP informs putative conformational epitopes,which determine the specific differences between the virus types,being valuable for epitope-focused immunogen design against ASFV infection.The minor capsid proteins form a complicated network below the outer capsid shell,stabilizing the capsid by holding adjacent capsomers together.Acting as core organizers,100-nm-length M1249 L proteins,running along each edge of the trisymmetrons and bridging two neighboring pentasymmtrons,form extensive intermolecular networks with other capsid proteins to guide the formation of capsid framework.These structural details unveil the basis of capsid stability and assembly,opening up new avenues for ASF vaccine development.
基金This work wassupported by the National Natural Science Foundation of China (Grant Nos. 39870174 and 39970155) the National "863" Project (Grant No. 103130306) and the National "973" Project (Grant Nos. G1999075602, G19990II902 and 1998051105).
文摘In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se-Met) derivative of TTR was overexpressed in E. coli strain M15, with pQE-30 plasmid, through IPTG induction in M9 medium containing Se-Met. The product was purified to an estimated homogeneity of greater than 95% according to SDS-PAGE, by a Ni-NTA metal affinity followed by a Mono Q anion exchange column chromatography. The successful substitution of Se-Met for methionine (Met) was confirmed by MALDI-TOF and ESI-Quadrupole Mass Spectrometry analysis. The derivative crystal was obtained using similar conditions as those for the native.
