Transglutaminase-mediated N- and C-terminal fluorescein labeling of a protein can support the native activity of the modified protein

Fluorescein and its analogs are among the best fluorophores to label proteins and the labeling generally involves chemical modification of a translated protein. Using this methodology, labeling at a specific position remains difficult. It is known that the guinea pig liver transglutaminase (TGase)-catalyzed enzymatic modification method can allow terminal-specific fluorophore labeling of a protein by monodansylcadaverine. However, native activity of the fluorescent protein has not been investigated so far, nor has direct comparison between the chemical modification and the TGase-catalyzed modification been attempted. Therefore, we compared the possibility of fluorescein labeling via chemical labeling and via TGase-catalyzed modification. The latter method was found to be very practical and overcame some of the problems associated with the specificity of the former; fluorescein was covalently attached only to the N- or C-terminal site of glutathione S-transferase when the reaction was catalyzed by TGase and the resulting labeled protein completely retained its native activity. The TGase-mediated labeling occurred not only at room temperature but also at 4 degrees C to the same extent, which is more desirable for preventing the inactivation of proteins.     

Prediction of the structures of proteins with the UNRES force field, including dynamic formation and breaking of disulfide bonds

The presence of disulfide bonds is essential for maintaining the structure and function of many proteins. The disulfide bonds are usually formed dynamically during folding. This process is not accounted for in present algorithms for protein-structure prediction, which either deduce the possible positions of disulfide bonds only after the structure is formed or assume fixed disulfide bonds during the course of simulated folding. In this work, the conformational space annealing (CSA) method and the UNRES united-residue force field were extended to treat dynamic formation of disulfide bonds. A harmonic potential is imposed on the distance between disulfide-bonded cysteine side-chain centroids to describe the energetics of bond distortion and an energy gain of 5.5 kcal/mol is added for disulfide-bond formation. Formation, breaking and rearrangement of disulfide bonds are included in the CSA search by introducing appropriate operations; the search can also be carried out with a fixed disulfide-bond arrangement. The algorithm was applied to four proteins: 1EI0 (alpha), 1NKL (alpha), 1L1I (beta-helix) and 1ED0 (alpha + beta). For 1EI0, a low-energy structure with correct fold was obtained both in the runs without and with disulfide bonds; however, it was obtained as the lowest in energy only with the native disulfide-bond arrangement. For the other proteins studied, structures with the correct fold were obtained as the lowest (1NKL and 1L1I) or low-energy structures (1ED0) only in runs with disulfide bonds, although the final disulfide-bond arrangement was non-native. The results demonstrate that, by including the possibility of formation of disulfide bonds, the predictive power of the UNRES force field is enhanced, even though the disulfide-bond potential introduced here rarely produces disulfide bonds in native positions. To the best of our knowledge, this is the first algorithm for energy-based prediction of the structure of disulfide-bonded proteins without any assumption as to the positions of native disulfides or human intervention. Directions for improving the potentials and the search method are suggested.     

浅谈现代塑料模具的设计与制造

随着现代塑料制品的形状越来越复杂，塑料模具的设计也越来越复杂。以载重汽车仪表板为例，介绍了鞠料模具设计的内容和过程。介绍了CAD／CAE／CAM、先进设备、手工加工、检测手段、反向工程、快速成型制造在模具制造中的应用。分析了现代塑料模具制造的发展方向和前景。     

A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system

Emulsion formulations used for in vitro compartmentalization (IVC) methods were found to be incompatible with protein expression in the rabbit reticulocyte (RRL) system, causing rapid discoloration and translation shutdown. Here we identify possible causes and describe a novel water-in-oil emulsion which abolished discoloration and allowed high-level in-emulsion expression of active luciferase and human telomerase using the RRL. This novel emulsion greatly expands the range of potential protein targets for IVC.     

Fusion of the antiferritin antibody VL domain to barnase results in enhanced solubility and altered pH stability

Chimeric immunotoxins that combine antigen recognition domains of antibodies and cytotoxic RNases have attracted much attention in recent years as potential targeted agents for cancer immunotherapy. In an attempt to obtain a structurally minimized immunofusion for folding/stability studies, we constructed the chimeric protein VL-barnase. The chimera comprises a small cytotoxic enzyme barnase, ribonuclease from Bacillus amyloliquefaciens, fused to the C-terminus of the light chain variable domain (VL) of the anti-human ferritin monoclonal antibody F11. While the individual VL domain was expressed in Escherichia coli as insoluble protein packed into inclusion bodies, its fusion to barnase resulted in a significant ( approximately 70%) fraction of soluble protein, with only a minor insoluble fraction ( approximately 30%) packed into inclusion bodies. The in vivo solubilizing effect of barnase was also observed in vitro and suggests a chaperone-like role that barnase exerted with regard to the N-terminal VL domain. Cytoplasmic VL-barnase was analyzed for structural and functional properties. The dimeric state of the chimeric protein was demonstrated by size-exclusion chromatography, thus indicating that fusion to barnase did not abrogate the intrinsic dimerization propensity of the VL domain. Ferritin-binding affinity and specificity in terms of constants of association with isoferritins were identical for the isolated VL domain and its barnase fusion, and RNase activity remained unchanged after the fusion. Intrinsic fluorescence spectra showed a fully compact tertiary structure of the fusion protein. However, significantly altered pH stability of the fusion protein versus individual VL and barnase was shown by the pH-induced changes in both intrinsic fluorescence and binding of ANS. Together, the results indicate that VL-barnase retained the antigen-binding affinity, specificity and RNase activity pertinent to the two individual constituents, and that their fusion into a single-chain chimeric protein resulted in an altered tertiary fold and pH stability.     

Zinc binding drives the folding and association of the homo-trimeric gamma-carbonic anhydrase from Methanosarcina thermophila

Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed beta-helical subunits of which bind three catalytic Zn(2+) ions at symmetry-related subunit interfaces. The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of approximately 70 kcal (mol trimer)(-1) at standard state. Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well described by a three-state model involving the folded apo-trimer, the folded monomer and the unfolded monomer. The monomer is estimated to have a stability of 4.0 +/- 0.3 kcal (mol monomer)(-1). The association to form apo-trimer contributes 13.2 +/- 0.4 kcal (mol trimer)(-1), a value confirmed by analytical ultracentrifugation measurements. Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer. The literature value for the free energy of binding of one Zn(2+) ion to a canonical active site, 16.4 kcal mol(-1), is consistent with the presumption that the >45 kcal (mol trimer)(-1) generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam.     

P507萃取法La／Ce、Ca／La分离工艺改进研究

稀土金属La、Ce分离过程中富集的Ca等非稀土金属采用传统的萃取分离和Ca／La在线分离工艺,只能将Ca降至0．05％,不能达到高纯氧化物的要求,文章根据相平衡原理,利用Ca^2＋、Mg^2＋、Zn^2＋等非稀土杂质与La^3＋在萃取剂中的萃取平衡差异,提出了P507—磺化煤油—HCl体系,La／Ce、Ca／La联合分离工艺,结果表明,新工艺可使LaCl3萃取液中的Ca^2＋与非稀土杂质含量降至50μg／g以下;使La2O3的收率提高到97％;而LaCl3的生产成本节约了20％,经济效益十分明显。     

抗坏血酸在锌铁合金镀液中的作用

本文讨论了抗坏血酸的氧化还原反应以及对锌铁合金镀液中Fe^2 和PH值的稳定作用。研究结果表明，抗坏血酸是一种良好的还原剂，虽然在锌铁合金镀液中用量仅为1－2g/l，但作用显著。     

Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization

Engineering the specificity of DNA-modifying enzymes has proven extremely challenging, as sequence recognition by these enzymes is poorly understood. Here we used directed evolution to generate a variant of HaeIII methyltransferase that efficiently methylates a novel target site. M.HaeIII methylates the internal cytosine of the canonical sequence GGCC, but there is promiscuous methylation of a variety of non-canonical sites, notably AGCC, at a reduced rate. Using in vitro compartmentalization (IVC), libraries of M.HaeIII genes were selected for the ability to efficiently methylate AGCC. A two-step mutagenesis strategy, involving initial randomization of DNA-contacting residues followed by randomization of the loop that lies behind these residues, yielded a mutant with a 670-fold improvement in catalytic efficiency (k(cat)/K(m)(DNA)) using AGCC and a preference for AGCC over GGCC. The mutant methylates three sites efficiently (AGCC, CGCC and GGCC). Indeed, it methylates CGCC slightly more efficiently than AGCC. However, the mutant discriminates against other non-canonical sites, including TGCC, as effectively as the wild-type enzyme. This study provides a rare example of a laboratory-evolved enzyme whose catalytic efficiency surpasses that of the wild-type enzyme with the principal substrate.     

A cell-based screen for function of the four-helix bundle protein Rop: a new tool for combinatorial experiments in biophysics

Combinatorial methodologies have revolutionized studies in biomolecular function, but they have so far proven less useful for understanding macromolecular structure and stability. This is largely because of the difficulty of screening libraries of molecules for biophysical properties, and the difficulty of interpreting structural effects in complicated molecules. Here, we report a novel, robust, cell-based screen for function of the four-helix bundle protein, Rop. By expression of green fluorescent protein from a ColE1 plasmid, the screen reports the copy number of the plasmid, which is modulated in Escherichia coli by Rop. We have engineered the screen so that the fluorescent phenotype can correspond to either Rop activity or lack thereof. We have used the screen to demonstrate with systematically constructed Rop core variants that not all molecules that bind small stem-loop RNAs in vitro are active in vivo. Rop is well understood from structural work and systematic mutations, which makes it possible to construct rational, targeted libraries. This screen makes it possible to rapidly interrogate such libraries effectively for proper protein folding and stability. In addition to its intended utility for combinatorial experiments in biophysics, the screen will allow further dissection of the mechanism of Rop-mediated plasmid copy number regulation in vivo.     

脂肪醇制备叔胺的新型催化剂的最新进展

对采用脂肪醇和二甲胺一步胺化制备(得率100%)相应的叔胺(烷基二甲基叔胺)的新型催化剂的最新进展进行了论述,对高活性Cu/Ni/Ca/Ba-St胶体催化剂(Cu0/Ni0/Ca0的比为5∶1∶1)和不含镍的Cu/La2O3进行了讨论。由此得出结论:要达到定量制备的关键因素是,通过结合还原钙或更活泼的促进剂,如Ce0和La0,尽可能地增加催化体系的氢解活性,由此形成金属氢化物,在铜/镍的作用下产生活性氢的“自供体系”。这一就地形成金属氢化物的基本功能将可极大地减少催化剂的用量,使铜质量分数减少到2×10-3%,从而极大地降低二甲胺的歧化,增加目的叔胺的选择性。总的来说,观察到在铜/镍固体催化剂以及胶体催化剂作用下,活性氢自供体系的基本功能使充入的大量氢一点都不被消耗掉。     

Effect of the Cobalt Ion and Polyethyleneimine Adsorption on the Surface Forces between Tungsten Oxide and Cobalt Oxide in Aqueous Media

We used the colloidal probe technique with atomic force microscopy to study the interactions between a tungsten oxide sphere, WO₃, and flat oxidized tungsten and cobalt surfaces in aqueous electrolytes. We investigated the effects of adsorption of cobalt ions to tungsten oxide surfaces and adsorption of polyethyleneimine (PEI). Low concentrations of cobalt ions added to a WO₃ system resulted in extended hydration forces and a lowering of the absolute value of the surface potential. PEI was shown to adsorb to the WO₃ surfaces and induce an electrosteric repulsion in both the symmetric (WO₃–WO₃) and asymmetric (WO₃–CoOOH) systems. The possible complexation of cobalt ions with PEI does not significantly influence the thickness of the adsorbed layer.     

Conservation and covariance in PH domain sequences: physicochemical profile and information theoretical analysis of XLA-causing mutations in the Btk PH domain

Mutations that cause X-linked agammaglobulinemia (XLA) appear throughout the Bruton tyrosine kinase (Btk) sequence, including the pleckstrin homology (PH) domain. To analyze the basis of this disease with respect to protein structure, we studied the relationships between PH domain sequences and structures by comparing sequence-based profiles of physicochemical properties and solvent accessibility profiles. The diversity of the distribution of amino acids was measured by calculating entropies for sequences containing mutations at different positions in multiple sequence alignments. Mutual information was calculated to quantify positional covariation. Eight conserved extrema were apparent in all profiles. The majority of the XLA disease-causing mutations in the Btk PH domain were found at positions having significant mutual information, indicating that there are covariant constraints for both structure and function. Together with additional structural analyses, all the XLA mutations that were analyzed could be explained at the molecular level. The method developed here is applicable to the design of mutations for protein engineering.     

LLDPE／EVA／CB复合导电体系电阻稳定性研究

以线性低密度聚乙烯（ＬＬＤＰＥ）和乙烯－醋酸乙烯共聚物（ＥＶＡ）为基体，油炉法炭黑（ＣＢ）为导电性赋与剂，探讨了热塑性半结晶性高聚物／炭黑复合体系的ＰＴＣ（正温度系数）效应及其电阻稳定性。结果表明，过氧化物交联剂（ＤＣＰ）可提高体系的ＰＴＣ性能稳定性和ＰＴＣ的转变温度，当用量为１％质量分数时，影响最为明显。炭黑接枝能提高体系的ＰＣＴ特性，比辐射更有效，使ＰＴＣ强度至少可提高１０倍。在所研究的极性高聚物Ａ、非极性高聚物Ｂ和极性物Ｃ三种接枝物中，以Ａ／ＰＥ／ＣＢ和Ｃ／ＰＥ／ＣＢ体系的ＰＴＣ特性明显，Ｂ／ＰＥ／ＣＢ体系峰值电阻率对应的温度低于Ａ／ＰＥ／ＣＢ约６～１５℃。在工作电压下通断电循环试验表明，ＬＬＤＰＥ／ＥＶＡ／Ａ－ｇ－ＣＢ体系在６０００ｈ的通电过程中，工作温度基本稳定在６５±５℃，通、断电循环试验的电阻率变化Ｒｆ／Ｒｉ大于２，功率变化Ｐｆ／Ｐｉ在１左右，而ＬＬＤＰＥ／ＥＶＡ／ＣＢ体系的发热温度下降很快，仅在７２０ｈ内就由８５℃下降到５０℃，直到３０℃才趋稳定，其电阻率增加３０倍以上，发热功率由１８Ｗ／ｍ下降到６Ｗ／ｍ。     

The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence

To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; lambda(max) = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; lambda(max) = 623 nm) red light-emitting luciferases. The emission spectrum (lambda(max) = 571 nm) and K(M) for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 220-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (lambda(max) = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.     

Band Gap Modification of Diamond Photonic Crystals by Changing the Volume Fraction of the Dielectric Lattice

Photonic crystals with a diamond structure of epoxy lattices in which TiO₂-based ceramic particles are dispersed were fabricated by stereolithography. The periodicity of the lattice was designed to reflect electromagnetic waves in the gigahertz range. The volume fraction (β) of the dielectric lattice medium was modified from 14% to 33% by changing the rod diameter of the lattice. The photonic band gap was observed along Γ-L 〈111〉, Γ-X 〈100〉, and Γ-K 〈110〉 directions and the complete photonic band gap was formed at over β= 20%. The width of the forbidden gap increased gradually when the β increased over 14%, and reached 2.4 GHz at β= 33%. These results agreed with the band calculation using the plane wave expansion method.     

高分子材料加工课程教学改革与实践

通过建立两种教学见习基地、编写教材、建立教学网站、进一步更新实验内容和增加开放性综合设计性项目、利用Pro/E、CAD等软件进行塑料成型工艺没计和仿真成犁实验等,实施具有地方产业特色的高分子材料加工课程改革.     

ABS/SAN/SMA三元合金性能研究

研究了ABS SAN SMA共混物。确定了配比和共混物的耐热性能、冲击性能、拉伸性能、断裂伸长率、模量、熔融指数等的关系     

流变法与DSC法测量PVC凝胶度的原理与结果讨论

本文阐明了用零长毛细管流变仪和差示扫描量热计测定ＰＶＣ熔体凝胶度的原理，并用这两种方法测量了ＰＶＣ／ＣＰＥ／ＰＳ体系的凝胶度。讨论了凝胶度与加工温度及力学性能的关系，并对这两种测试结果进行了比较。     

Expression of the phosphorylase kinase {gamma} subunit catalytic domain in Escherichia coli

The catalytic subunit of phosphorylase b kinase () and an engineeredtruncated form (-trc, residues 1 –297) have been expressedin Escherichia coli. The truncated protein included the entirecatalytic domain as defined by sequence alignment with otherprotein kinases but lacked the putative calmodulin binding domain.Full-length protein was produced in insoluble aggregates. Someactivity was regenerated by solubilization in urea and dilutioninto renaturating buffer but the activity was found to be associatedwith a smaller molecular weight component. Full-length proteincould not be refolded successfully. The truncated subunit wasproduced in the soluble fraction of the cell as well as in inclusionbodies. The insoluble protein was refolded by dilution fromurea and purified to homogeneity, in a one step separation onDEAESepharose to give a protein mol. wt 32 000 ± 2000with a high sp. act. of 5.3 µmol ³²p incorporated intophosphorylase b(PPB)/min/nmol. Kinetic parameters gave K_m forATP 46 ± 3 µM and K_m, for PPb 27 ± 1 µM.The sp. act. and the Am values are comparable to those observedfor the activated holoenzyme and indicate that the -trc retainsthe substrate recognition and catalytic properties. The ratioof activities at pH 6.8/8.2 was 0.84. -trc was inhibited byADP with a K_i of 52 µM and was sensitive to activationby Mg²⁺ and inhibition by Mn²⁺, properties that are characteristicof the holoenzyme and the isolated subunit. Calmodulin whichconfers calcium sensitivity on the isolated subunit had noeffect on the enzymic properties of -trc. A small inhibitionby free Ca²⁺ was observed with peptide substrate.