Purification of clathrin assembly protein from rat liver

Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.     

Molecular cloning of clathrin assembly protein gene (rCALM) and its differential expression to AP180 in rat brain

Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicle (CCV) in neurons. The clathrin assembly protein gene (rCALM) was cloned from rat brain cDNA library. rCALM deduced 69 kD molecule has overall 73% amino acid homology compared with that of AP180 protein. The N-terminal domain, where amino acid sequences are very similar with AP180, harbours binding sites for clathrin and inositides, as well as possible phosphorylation sites, but the proline rich C-terminal domain is different from that of AP180. The mRNA expression of rCALM and AP180 by in situ hybridization histochemistry revealed that the rCALM mRNA was more intensely expressed than that of AP180, and the distribution patterns were different from each other. These results suggest that the rCALM mediates the assembly of clathrin in neural and supporting cells of brain, and regulates the clathrin coated-vesicle formation through phosphorylation and inositide metabolism.     

Assembly of Clathrin Baskets

The creation and internalization of small, protein-coated vesicles is a central factor in the uptake of materials from the surfaces of eucaryotic cells through a process known as receptor mediated endocytosis. Under appropriate in vitro conditions, the principal coat component, viz., clathrin, which is found in the cell as a trimer joined at a common hub, assembles into polyhedral ‘baskets’ (‘cages’) containing pentagonal and hexagonal facets. These reconstituted cages have the appearance of miniscule soccer-ball like structures. Their sizes vary over a finite range whose limits depend on the presence or absence of ancillary proteins (‘assembly proteins’) that are known to increase the tendency for the baskets to form. By fitting data on basket size distributions to simple energetic (thermodynamic) models, one is able to estimate mechanical properties of the clathrin constituents of the baskets and infer the role of assembly proteins in strengthening interactions between the clathrin components of the struts that constitute the edges of the baskets.     

Protein Delivery System Containing a Nickel‐Immobilized Polymer for Multimerization of Affinity‐Purified His‐Tagged Proteins Enhances Cytosolic Transfer

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea‐grafted polyethylenimine (πPEI) with affinity‐purified His‐tagged proteins pre‐organized onto a nickel‐immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His‐tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His‐tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single‐chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.     

Structure of the EntB multidomain nonribosomal peptide synthetase and functional analysis of its interaction with the EntE adenylation domain

Nonribosomal peptide synthetases are modular proteins that operate in an assembly line fashion to bind, modify, and link amino acids. In the E. coli enterobactin NRPS system, the EntE adenylation domain catalyzes the transfer of a molecule of 2,3-dihydroxybenzoic acid to the pantetheine cofactor of EntB. We present here the crystal structure of the EntB protein that contains an N-terminal isochorismate lyase domain that functions in the synthesis of 2,3-dihydroxybenzoate and a C-terminal carrier protein domain. Functional analysis showed that the EntB-EntE interaction was surprisingly tolerant of a number of point mutations on the surface of EntB and EntE. Mutational studies on EntE support our previous hypothesis that members of the adenylate-forming family of enzymes adopt two distinct conformations to catalyze the two-step reactions.     

人三叶因子3双结构域突变体的构建

利用基因工程方法在大肠杆菌-酶母穿梭质粒pPIC9K上构建了人三叶因子3(HumanTrefoilFactor3,hTFF3)双结构域突奕体基因,采用毕氏酵母表达系统对目的基因进行了分泌表达。经S-Sepharose,Q-Sepharose,SephacrylS-100纯化后得到突变体蛋白,主要以单体形式存在。SDS-PAGE表明重组蛋白分子量约为12000,并在Westernblotting印迹实验中证明能被抗hTFF3抗体所识别,N-端氨基酸测序与天然hTFF3-一致,质谱检测纯化的蛋白出现单体和双体两种形式。用重组蛋白对乙醇诱导的大鼠胃溃疡进行实验治疗表明hTFF3双体突变体与天然形成的双体蛋白具有相同的活力,并且活力高于单体形式的hTFF3。     

Accelerated screening of phage-display output with alkaline phosphatase fusions

When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin N-terminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.     

具有生物活性的人带3蛋白胞质片段融合蛋白的克隆、表达与纯化

用PCR法从质粒pHB3中扩增了人红细胞带3蛋白胞质片段(CDB3)基因.PCR产物经限制性内切酶切割后与多克隆位点处带有编码6个组氨酸序列的高效表达载体pET28b连接,构建为重组子pCDBHistag.重组子经酶切及序列测定后在大肠杆菌BL21(DE3)中获得高效表达,可溶性目的蛋白占菌体总蛋白的40%左右.C端带有6个连续组氨酸的带3蛋白胞质片段作为融合蛋白不仅可以降低宿主菌蛋白酶对其水解程度,而且简化了目的蛋白的纯化过程.经一步螯合Ni²⁺的亲和层析获得了电泳纯的带3蛋白胞质片段融合蛋白.活性测定结果表明,带3蛋白胞质片段融合蛋白能够抑制醛缩酶(Aldolase)活性的70%,与文献报道的人红细胞内带3蛋白胞质片段具有相同的功能.     

人野生型MBL基因在大肠杆菌中的表达

为获得人MBL蛋白，并对其功能进行初步研究，用DNA重组法构建了组氨到标签融合原核表达质粒pET28(b)-MBL。将重组质粒转入大肠杆菌BL21（DE3），经IPTG在37℃条件下诱导培养，利用SDS－PAGE，Westem-blot检测目的蛋白的表达，用IMAC金属螯合层析柱对其进行纯化。成功地表达了重组MBL蛋白，纯化的MBL浓度约为844μg/mL，为制备MBL的基因工程抗体奠定了基础。     

小麦ACCase CT功能域基因在大肠杆菌中的表达及与除草剂的相互作用

以中国春小麦幼苗为材料,克隆构建了小麦质体乙酰辅酶A羧化酶(ACCase)的羧基转移酶(CT)重组质粒( RCP18-5),并实现了重组质粒在大肠杆菌中的可溶性高表达.对重组蛋白的性质研究表明,该蛋白具有较强的疏水性,稳定性不高.为改善这种状况,对CT功能域基因进行了截短和延长,同样于大肠杆菌中进行表达.结果表明,仅长...     

Template engineering through epitope recognition: a modular, biomimetic strategy for inorganic nanomaterial synthesis

Natural systems often utilize a single protein to perform multiple functions. Control over functional specificity is achieved through interactions with other proteins at well-defined epitope binding sites to form a variety of functional coassemblies. Inspired by the biological use of epitope recognition to perform diverse yet specific functions, we present a Template Engineering Through Epitope Recognition (TEThER) strategy that takes advantage of noncovalent, molecular recognition to achieve functional versatility from a single protein template. Engineered TEThER peptides span the biologic-inorganic interface and serve as molecular bridges between epitope binding sites on protein templates and selected inorganic materials in a localized, specific, and versatile manner. TEThER peptides are bifunctional sequences designed to noncovalently bind to the protein scaffold and to serve as nucleation sites for inorganic materials. Specifically, we functionalized identical clathrin protein cages through coassembly with designer TEThER peptides to achieve three diverse functions: the bioenabled synthesis of anatase titanium dioxide, cobalt oxide, and gold nanoparticles in aqueous solvents at room temperature and ambient pressure. Compared with previous demonstrations of site-specific inorganic biotemplating, the TEThER strategy relies solely on defined, noncovalent interactions without requiring any genetic or chemical modifications to the biomacromolecular template. Therefore, this general strategy represents a mix-and-match, biomimetic approach that can be broadly applied to other protein templates to achieve versatile and site-specific heteroassemblies of nanoscale biologic-inorganic complexes.     

Assembly of the hexameric Escherichia coli arginine repressor investigated by nano-electrospray ionization time-of-flight mass spectrometry

The arginine repressor (ArgR) from Escherichia coli regulates genes for L-arginine metabolism and is a required recombination factor for colE1 plasmid replication. Both functions require binding of L-arginine to the protein. In this work, nano-electrospray ionization time-of-flight mass spectrometry (nano-ESI-TOFMS) is used to study conformational and oligomeric states of intact ArgR and its isolated structural domains. In agreement with X-ray diffraction studies, it is shown that ArgR oligomerizes to form hexamers in both the presence and absence of L-arginine, and the basic unit of oligomerization appears to be the trimer. Higher-order assembly into dodecamers is also detected. The isolated C-terminal domain is found to associate into trimers and hexamers whereas the N-terminal domain is detected in its monomeric form. The observed species distributions suggest a role for the N-terminal domain in hexamer stabilization.     

乙型肝炎病毒包膜M蛋白的高效表达和分离纯化

从病人血清中经过聚合酶链反应扩增得到了编码HBV-M蛋白的基因片段(PreS2+S),将其转入原核表达载体pET-His,构建了原核表达质粒pET-His-M。重组质粒转化大肠杆菌BL21,IPTG诱导表达,收获菌体超声波破碎后,蔗糖梯度溶液洗脱杂质。HBV-M在原核系统中高效表达,分子量为30kD,纯化后得到了纯度为98%的HBV-M蛋白,ELISA检测结构表明M蛋白的抗原性比S蛋白的抗原性强。该研究为进一步研究HBV包膜M蛋白的结构、功能及新型乙肝疫苗的研制奠定了基础。     

Chemical glycosylation of cytochrome c improves physical and chemical protein stability

Background

Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability.

Results

Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac₄-Cyt-c, Lac₉-Cyt-c, Dex₅(10kD)-Cyt-c, Dex₈(10kD)-Cyt-c, and Dex₃(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86?±?3 to 95?±?1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92?±?7% to 96?±?4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex₃(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24?±?8% and 26?±?6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers.

Conclusion

The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.

     

重组HEV衣壳蛋白截短体的表达及鉴定

戊型肝炎(HE)是由戊型肝炎病毒(HEV)感染引起的肠道病毒性传染病. HEV是一种无囊膜的单股正链RNA病毒, 其编码区由3个开放阅读框(ORF)组成, 属戊型肝炎病毒科. HEV衣壳蛋白由ORF2编码. 本研究根据编码HEV ORF2 aa382~aa674的核苷酸序列克隆了p293基因, 并将其克隆入原核表达载体pET28a, 利用大肠杆菌BL21(DE3)对HEV衣壳蛋白截短体(p293)进行了表达. SDS-PAGE和Western blot检测结果表明, 在优化的表达条件下(1 mmol/L IPTG, 250 r/min, 37℃, 5 h), 重组蛋白p293能够在大肠杆菌内有效表达, 目的蛋白约占总蛋白的66.15%. TEM检测结果显示, 原核表达的p293能够在体外形成约30~40 nm的病毒样颗粒. 免疫印迹和免疫荧光检测结果表明, p293与HEV标准阳性血清具有良好的反应原性和反应特异性. 实验结果表明, p293可应用于HEV宿主吸附和病毒装配研究, 为HEV的预防与诊断研究奠定了基础.     

Multivalent-Interaction-Driven Assembly of Discrete,Flexible, and Asymmetric Supramolecular Protein Nano-Prisms

Current approaches to design monodisperse protein assemblies require rigid, tight, and symmetric interactions between oligomeric protein units. Herein, we introduce a new multivalent-interaction-driven assembly strategy that allows flexible, spaced, and asymmetric assembly between protein oligomers. We discovered that two polygonal protein oligomers (ranging from triangle to hexagon) dominantly form a discrete and stable two-layered protein prism nanostructure via multivalent interactions between fused binding pairs. We demonstrated that protein nano-prisms with long flexible peptide linkers (over 80 amino acids) between protein oligomer layers could be discretely formed. Oligomers with different structures could also be monodispersely assembled into two-layered but asymmetric protein nano-prisms. Furthermore, producing higher-order architectures with multiple oligomer layers, for example, 3-layered nano-prisms or nanotubes, was also feasible.     

Artificial polypeptide scaffold for protein immobilization

An artificial polypeptide scaffold composed of surface anchor and protein capture domains was designed and expressed in vivo. By using a mutant E. coli phenylalanyl-tRNA synthetase, the photoreactive amino acid para-azidophenylalanine was incorporated into the surface anchor domain. Octyltrichlorosilane-treated surfaces were functionalized with this polypeptide by spin coating and photocrosslinking. The resulting protein films were shown to immobilize recombinant proteins through association of coiled coil heterodimer.     

Boerhavia diffusa plant extract can be a new potent therapeutics against mutant nephrin protein responsible for type1 nephrotic syndrome: Insight into hydrate-ligand docking interactions and molecular dynamics simulation study

Nephrotic syndrome type 1 is an inherited condition in which mutation of NPHS1 gene, which encodes nephrin protein, results increase permeability of glomerular capillary wall for macromolecules causes heavy proteinuria, hypoproteinemia, and edema. Nephrotic syndrome patients are resistant to steroid and immunosuppressive treatment. Natural products have traditionally been used to treat a number of chronic diseases. Present study was focused to investigate potency of different phytochemicals found in Boerhavia diffusa (B.diffusa) plant against mutant nephrin protein. The study involves virtual screening of total 66 bioactive compounds from B.diffusa plant against wild type and mutant models of Ig4 domain of nephrin protein through AutoDock raccoon. Based on binding energy and drug-likeness property, seven phytocompounds were screened. Hydrate-ligand docking (addition of explicit waters in ligands) method was used to select potential phytocompounds that could bind to mutant model of Ig4 domain of nephrin protein. For further prediction, molecular dynamics simulations with 100ns trajectory of Ig4 domain of nephrin protein in glycolipid bilayer membrane for systems such as wild, mutant, mutant model complex with boeravinone M and mutant model complex with boeravinone E were thoroughly studied. Hydrate-ligand docking result predicted boeravinone M and boeravinone E have shown better binding performance with mutant model. It causes due to hydration force field that measures entropy and enthalpy for each water molecule separately, allowing for more exact estimations of their contribution to ligand-protein interaction. Boeravinone E shows lowest short-range Lennard-Jones (LJ-SR) interaction energies of ?47.78 ± 12.7 kJ/mol. The results showed reduced compactness and stability nature of mutant model of Ig4 domain of nephrin protein, however, binding with boeravinone E has effectively modulated its stability and function by increasing its compactness. Current study may provide insight into therapeutic development of boeravinone E as a potential inhibitor against NPHS1in near future.