GST-HRB融合蛋白的表达与纯化

构建GST-HRB重组质粒,进行融合蛋白的表达、纯化及鉴定.利用PCR扩增及基因重组技术,以pcDNA-3.1-HRB为模板扩增出HRB全基因序列,并将其插入带有GST(谷胱甘肽巯基转移酶)标签的原核表达载体pGEX-6P-1中,构建GST-HRB融合蛋白表达质粒.然后,将重组质粒GST-HRB转化至大肠杆菌Rosseta进行融合蛋白的表达.利用GST琼脂糖珠进行融合蛋白的纯化,最后应用SDS-PAGE电泳和Western blotting鉴定纯化的融合蛋白.结果表明,成功构建pGEX-6P-1-HRB原核表达载体,表达及纯化了GST-HRB融合蛋白.     

炭疽芽孢杆菌EA1蛋白的融合表达和纯化

目的：原核表达重组炭疽芽孢杆菌EA1蛋白。方法：用PCR方法从炭疽芽孢杆菌A16R疫苗株染色体中扩增编码EA1蛋白的eag基因序列,经过纯化、酶切后克隆到含有GST标签的原核表达载体pGEX-6P-2中,构建重组载体pGEX-EA1;将空载体（作为对照）、重组载体转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,对其表达和纯化条件进行优化;利用Western印迹检测融合蛋白的表达。结果：构建了EA1蛋白的融合表达载体,并在大肠杆菌中获得高效表达;经Glutathione Sepharose 4B纯化获得了EA1蛋白;Western印迹表明,此蛋白可与GST标签抗体反应。结论：在原核表达系统中表达并纯化得到EA1融合蛋白,为进一步对其进行功能研究奠定了基础。     

大肠杆菌ybfE基因的三种原核质粒表达水平的对比及蛋白纯化北大核心

[目的]构建大肠杆菌功能未知基因ybf E的pET16b、pET32a和p GEX-4T-1三种原核表达系统,通过对比表达水平筛选出最优表达体系,并纯化表达的可溶性Ybf E融合蛋白。[方法]使用pET16b、pET32a和p GEX-4T-1表达质粒构建pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达载体,分别转化大肠杆菌BL21,IPTG诱导表达Ybf E融合蛋白,对三种表达系统的表达水平进行对比,并对pET16b-ybf E和pGEX-4T-1-ybf E表达体系的裂菌上清中的可溶性Ybf E融合蛋白液分别使用镍柱和GST蛋白纯化柱纯化。[结果]构建了pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达体系,并使用IPTG诱导表达Ybf E融合蛋白。ybf E在p GEX-4T-1载体内的表达水平最高,接下来依次为pET16b和pET32a。pET16b-ybf E和p GEX-4T-1-ybf E表达的可溶性Ybf E融合蛋白纯化后浓度分别为86μg/m L和724μg/m L。[结论]成功构建了ybf E基因的三种原核表达系统,筛选出最佳表达体系,可溶性Ybf E融合蛋白得到纯化。     

大肠杆菌ybfE基因的三种原核质粒表达水平的对比及蛋白纯化

[目的]构建大肠杆菌功能未知基因ybf E的pET16b、pET32a和p GEX-4T-1三种原核表达系统,通过对比表达水平筛选出最优表达体系,并纯化表达的可溶性Ybf E融合蛋白。[方法]使用pET16b、pET32a和p GEX-4T-1表达质粒构建pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达载体,分别转化大肠杆菌BL21,IPTG诱导表达Ybf E融合蛋白,对三种表达系统的表达水平进行对比,并对pET16b-ybf E和pGEX-4T-1-ybf E表达体系的裂菌上清中的可溶性Ybf E融合蛋白液分别使用镍柱和GST蛋白纯化柱纯化。[结果]构建了pET16b-ybf E、pET32a-ybf E和p GEX-4T-1-ybf E原核表达体系,并使用IPTG诱导表达Ybf E融合蛋白。ybf E在p GEX-4T-1载体内的表达水平最高,接下来依次为pET16b和pET32a。pET16b-ybf E和p GEX-4T-1-ybf E表达的可溶性Ybf E融合蛋白纯化后浓度分别为86μg/m L和724μg/m L。[结论]成功构建了ybf E基因的三种原核表达系统,筛选出最佳表达体系,可溶性Ybf E融合蛋白得到纯化。     

鲎血细胞中脂多糖结合蛋白TALF在大肠杆菌中的表达研究

TALF(Tachyleus antilipoposaccharide factor)对细菌内毒素（LPS）的核心部分有抑制作用。研究TALF cDNA基因在大肠杆菌中的表达,首先将TALF cDNA基因分别插入大肠杆菌表达载体pGEX-4T-2、pET22b、pET28a中,构建重组表达质粒,转化于大肠杆菌BL21(DE3)。结果表明克隆于pET22b、pET28a中的TALF cDNA基因没有表达,而融合了GST的TALF基因（GST-TALF）能够在大肠杆菌中表达,并形成包涵体。从1L培养基中可获得4mg纯度为91%的GST-TALF融合蛋白。经复性和纯化后的融合蛋白GST-TALF几乎检测不到抑菌活性及LPS中和活性,但该融合蛋白经凝血酶消化后表现出明显的体外抑菌活性及LPS中和活性。     

GST pull-down验证流感病毒PB1-F2蛋白与热休克蛋白40的相互作用

为体外验证流感病毒PB1-F2与热休克蛋白Hsp40相互作用,通过两个方向的GST pull-down试验验证PB1-F2与Hsp40的相互作用。构建GST-多肽融合蛋白原核表达载体pGEX-6P-1-PB1-F2和pGEX-6P-1-Hsp40,并在大肠杆菌(E.co-li)BL21中诱导表达;构建真核表达载体pLEGFP-Hsp40及pCAGGS-PB1-F2,并分别转染293T细胞使其表达Hsp40及PB1-F2融合蛋白,然后进行GST pull-down试验验证二者的相互作用。成功地构建了两种蛋白的各种表达载体,经表达、纯化获得了可溶性的GST-多肽融合蛋白,GST pull-down试验正反两方向都证实了PB1-F2与Hsp40的相互作用,初步证实了流感病毒PB1-F2在体外能与Hsp40发生相互作用。     

HIV-1Gag蛋白在大肠杆菌和重组杆状病毒系统中的高效表达及通用型温控原核表达载体的构建

将中国株HIV－1B亚型的gag全基因序列,克隆到杆状病毒表达载体pfastbacI中,构建了重组质粒pfastGag,利用细菌／杆状病毒表达系统筛选重组杆状病毒,在昆虫细胞中高效表达了HIV－1Gag蛋白。通过改造原核表达载体pBV220和pET28,构建了一种新的通用型温控原核表达载体质粒pVV5,该载体携带PrPl串联温控启功子及His—Tag纯化标签,利于目的蛋白表达与纯化。将HIV－1gag基因的1148一1857编码序列,分别插入到pVV5b、pET28b的相应位点,构建了重组表达质粒pEG1b、pEG7b,二者在不同受体菌中,表达重组蛋白的量分别占全菌体蛋白总量的42％和28％。利用IMAC金属螯合层析柱,对包涵体中的重组p24蛋白进行纯化,纯度超过80％;纯化后的重组蛋白可与HIV－1型标准阳性血清发生较强的免疫学反应。     

GST—talinl融合蛋白的原核表达及纯化

应用PCR方法扩增talinl的cDNA,并将其重组入谷胱甘肽转硫酶融合基因表达载体pGEX-4T-1中,获取人源的GST—talinl融合蛋白,为下阶段深入的研究talinl的结构、功能、及其与之相互作用的蛋白打下基础。经酶切、序列鉴定．选择正确重组子,将其质粒转化大肠杆菌BL21（DE3）,IPTG诱导表达,用Glutathione Sepharose 4B柱纯化,western blot鉴定。克隆得到了一个2400bp的talinl的cDNA片断,重组质粒目的DNA测序正确,纯化出分子量约为121、6kD的融合蛋白。用基因工程方法使GST—talinl重组质粒在原核细胞表达并成功纯化出GST—talinl融合蛋白。     

家蝇幼虫抗菌肽Attacin基因的克隆表达及抑菌生物学活性

目的克隆家蝇幼虫Attacin抗菌肽基因．构建原核融合表达载体,建立Attacin体内抗菌活性检测系统,优化表达和纯化Attacin目的蛋白,并初步研究其抗菌生物学功能。方法以pUC m-T／Attacin重组质粒为模板,设计特异性引物,PCR扩增Attacin编码区序列,分别克隆至原核表达载体pET30a（＋）和pGEX-4T-1。构建原核重组质粒,转化大肠埃希菌,表达重组Attacin蛋白,并在大肠埃希菌中体内检测Attacin的抗菌活性。利用亲和层析柱纯化重组融合蛋白Attacin,SDS-PAGE进行纯度分析,琼脂糖平板抑菌试验鉴定其生物活性。结果pET30a（a＋）／Attacin和pGEX-4T—1／Attacin重组质粒分别转化大肠埃希菌后,以IPTG诱导表达,与未诱导对照相比,含有重组质粒的宿主菌生长受到抑制。从pET30a（＋）／Attacin重组质粒的表达宿主菌中未能获得His-Attacin融合蛋白,而从pGEX-4T—1／Attacin重组质粒转化菌种获得GST-Attacin融合蛋白。SDS-PAGE分析表明Attacin重组蛋白分子量与预期结果一致,琼脂糖平板抑菌试验显示重组Attacin具有抗菌活性。结论Attacin基因在原核系统中成功表达,并且纯化后具有抑菌活性,为下一步研究Attacin的生物学功能及其应用开发奠定了基础。     

人Hepassocin的原核可溶性表达与纯化

目的：构建人Hepassocin的原核表达载体,可溶性表达并纯化得到高纯度的重组人Hepassocino方法：将人Hepassocin基因克隆到原核表达载体pET40b（＋）,转化大肠杆菌BL21（DE3）,于28℃经0．1mmol／LIPTG诱导6h,表达Ds-bC-Hepassocin融合蛋白,经镍柱纯化可溶性融合蛋白,用肠激酶切除融合蛋白的DsbC-His标签,再用镍柱纯化分离酶切后的Hepassocin,通过超滤进一步纯化并浓缩,用Western blot验证纯化后的Hepassocin。结果：构建了pET40b-Hepassocin原核表达载体,经诱导表达、亲和层析和肠激酶切除融合标签,获得了相对分子质量约32000的可溶性高纯度蛋白,Western blot鉴定证实该蛋白为不含融合标签的重组人Hepassocin。结论：实现了人Hepassocin的原核可溶性表达,通过纯化获得了较高纯度的重组人Hepassocin,为制备其单克隆抗体,进一步研究其生物学功能奠定了基础。     

Central Region of Talin Has a Unique Fold That Binds Vinculin and Actin

Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359–1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.     

Structural and biophysical analysis of BST-2/tetherin ectodomains reveals an evolutionary conserved design to inhibit virus release

BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-Å crystal structure of the complete mouse BST-2 ectodomain reveals an ∼145-Å parallel dimer in an extended α-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 Å for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.     

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-βGlcA-(1,4)- αGlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, single-stranded parallel β-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel β-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-β-elimination site of heparinase II from Pedobacter heparinus.     

Structures important in mammalian 11β- and 17β-hydroxysteroid dehydrogenases

We have used the X-ray crystallographic structures of rat and human dihydropteridine reductase and Streptomyces hydrogenans 20β-hydroxysteroid dehydrogenase to model parts of the 3-dimensional structure of human 11β- and 17β-hydroxysteroid dehydrogenases. We use this information along with previous results from studies of Drosophila alcohol dehydrogenase mutants to analyze the structures in binding sites for NAD(H) and NADP(H) in 11β-hydroxysteroid dehydrogenase-types 1 and 2. We also examine the structure of an -helix at catalytic site of 17β-hydroxysteroid dehydrogenase-types 1, 2, 3, and 4. This -helix contains a highly conserved tyrosine and lysine. Adjacent to the carboxyl side of this lysine is a site proposed to be important in subunit association. We find that 11β- and 17β-hydroxysteroid dehydrogenases-type 1 have the same residues at the “anchor site” and conserve other stabilizing features, despite only 20% sequence identity between their entire sequences. Similar conservation of stabilizing structures is found in the 11β- and 17β-hydroxysteroid dehydrogenases-type 2. We suggest that interactions of the dimerization surface of -helix F with proteins or membranes may be important in regulating activity of hydroxysteroid dehydrogenases.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.     

Trypanosoma cruzi macrophage infectivity potentiator has a rotamase core and a highly exposed α-helix

The macrophage infectivity potentiator protein from Trypanosoma cruzi (TcMIP) is a major virulence factor secreted by the etiological agent of Chagas’ disease. It is functionally involved in host cell invasion. We have determined the three-dimensional crystal structure of TcMIP at 1.7 Å resolution. The monomeric protein displays a peptidyl-prolyl cis–trans isomerase (PPIase) core, encompassing the characteristic rotamase hydrophobic active site, thus explaining the strong inhibition of TcMIP by the immunosuppressant FK506 and related drugs. In TcMIP, the twisted β-sheet of the core is extended by an extra β-strand, preceded by a long, exposed N-terminal α-helix, which might be a target recognition element. An invasion assay shows that the MIP protein from Legionella pneumophila (LpMIP), which has an equivalent N-terminal α-helix, can substitute for TcMIP. An additional exposed α-helix, this one unique to TcMIP, is located in the C-terminus of the protein. The high-resolution structure reported here opens the possibility for the design of new inhibitory drugs that might be useful for the clinical treatment of American trypanosomiasis.     

Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1^G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.     

The Common Structural Architecture of Shigella flexneri and Salmonella typhimurium Type Three Secretion Needles

The Type Three Secretion System (T3SS), or injectisome, is a macromolecular infection machinery present in many pathogenic Gram-negative bacteria. It consists of a basal body, anchored in both bacterial membranes, and a hollow needle through which effector proteins are delivered into the target host cell. Two different architectures of the T3SS needle have been previously proposed. First, an atomic model of the Salmonella typhimurium needle was generated from solid-state NMR data. The needle subunit protein, PrgI, comprises a rigid-extended N-terminal segment and a helix-loop-helix motif with the N-terminus located on the outside face of the needle. Second, a model of the Shigella flexneri needle was generated from a high-resolution 7.7-Å cryo-electron microscopy density map. The subunit protein, MxiH, contains an N-terminal α-helix, a loop, another α-helix, a 14-residue-long β-hairpin (Q51–Q64) and a C-terminal α-helix, with the N-terminus facing inward to the lumen of the needle. In the current study, we carried out solid-state NMR measurements of wild-type Shigella flexneri needles polymerized in vitro and identified the following secondary structure elements for MxiH: a rigid-extended N-terminal segment (S2-T11), an α-helix (L12-A38), a loop (E39-P44) and a C-terminal α-helix (Q45-R83). Using immunogold labeling in vitro and in vivo on functional needles, we located the N-terminus of MxiH subunits on the exterior of the assembly, consistent with evolutionary sequence conservation patterns and mutagenesis data. We generated a homology model of Shigella flexneri needles compatible with both experimental data: the MxiH solid-state NMR chemical shifts and the state-of-the-art cryoEM density map. These results corroborate the solid-state NMR structure previously solved for Salmonella typhimurium PrgI needles and establish that Shigella flexneri and Salmonella typhimurium subunit proteins adopt a conserved structure and orientation in their assembled state. Our study reveals a common structural architecture of T3SS needles, essential to understand T3SS-mediated infection and develop treatments.     

The secondary structure content of pigment-protein complexes from the thylakoids of two Chromophyte algae

The light-harvesting complex and Photosystem I have been isolated from thylakoids of two chromophyte algae by digitonin solubilisation and sucrose-gradient centrifugation. The -helic and β-structure content was determined by ultraviolet circular dichroism. The values obtained were approx. 40% -helix and approx. 14% β-structure for the light-harvesting complex of both Pavlova lutherii and Phaeodactylum tricornutum. For Photosystem I the values were approx. 55% -helix and 7% β-structure for both algae. It is concluded that for all photosynthetic antennae containing chlorophyll the dominant secondary structure is -helix.