碱性蛋白酶工程菌发酵条件及重组酶的纯化和性质的研究

在5L发酵罐中对重组碱性蛋白酶工程菌株BP071高产碱性蛋白酶的条件进行了研究,通过提高通气量和改变搅拌转速,BP071可在发酵40 h内达到产酶高峰,酶活力最高可达24480 u/mL。利用快速蛋白液相层析（FPLC）技术,建立了快速高效纯化碱性蛋白酶的方案。发酵液通过硫酸铵沉淀、DEAE-A-50脱色及聚乙二醇浓缩得粗酶,再经过CM-Sephadex-C-50、Sephadex-G-75柱层析后得到了单一组份的重组碱性蛋白酶,酶纯度提高了76.2倍。SDS-PAGE显示重组碱性蛋白酶分子量为28 kD。酶学性质研究表明,酶的最适作用pH为11,最适作用温度为60℃,具有良好的pH稳定性和热稳定性。Ca²⁺、Mg²⁺对酶的稳定性有促进作用,Hg²⁺、Ag⁺、PMFS和DFP能强烈抑制酶的活力。SDS和Urea对酶的活力无影响。     

The ReH6[P(C6H5)3]2 ion as a ligand: complexes with M(CO)3 fragments (M = Cr, Mo, W)

The reactions of [(H₅C₆)₃P]₂ReH₆⁻ with (CH₃CN)₃Cr(CO)₃, (diglyme)Mo(CO)₃ or (C₃H₇CN)₃W(CO)₃ led to the formation of [(H₅C₆)₃P]₂ReH₆M(CO)₃⁻ (M = Cr, Mo, W) complexes. These have been characterized by IR and NMR spectroscopies, as well as elemental analyses. A single crystal X-ray diffraction study has also been carried out for the M = Cr complex as a K(18-crown-6)⁺ salt. The complex crystallizes as a THF monosolvate in the monoclinic space group P2₁/n with a = 22.323(6), B = 9.523(2), C = 27.502(5) Å, β = 104.98(2)⁰ and V = 5648 ų for Z = 4. The Re---Cr separation is 2.5745(12) Å, and the two phosphine ligands are oriented unsymmetrically. Although the hydride ligands were not found, the presence of three bridging hydrides and a dodecahedral coordination geometry about rhenium could be inferred. Low temperature ¹H and ³¹P NMR spectroscopic studies did not reveal the low symmetry of the solid state structure.     

Electronic spectra of the chromium chalcocarbonyl complexes,Cr(CO)5(CX) and (η6-C6H6)Cr(CO)2(CX) (X= O,S,Se)

The room-temperature electronic spectra of the chromium chalcocarbonyl complexes, Cr(CO)₅(CX) and (η⁶-C₆H₆)Cr(CO)₂(CX) (X = O, S, Se), have been recorded in solution, and in some cases, in the gas phase. Assignments for the thiocarbonyl and selenocarbonyl spectra are proposed on the basis of the literature assignments for the parent all-CO derivatives. Overall, the data support the order of increasing electron withdrawing capacity of the chalcocarbonyl ligands as CO < CS < CSe.     

Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme

Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.     

Syntheses and structures of the heterometallic clusters [(Ph3PAu)3Re(CO)4], [(Ph3PAu)4Re(CO)4], [(Ph3PAu)6AuRe2(CO)8], and [(Ph3PAu)6Re(CO)3]

The reaction of [HRe₃(CO)₁₂]²⁻ with an excess of Ph₃PAuCl in CH₂Cl₂ yields [(Ph₃PAu)₄Re(CO)₄]⁺ as the main product, which crystallizes as [(Ph₃PAu)₄Re(CO)₄]PF₆ · CH₂Cl₂ (1 · CH₂Cl₂) after the addition of KPF₆.The crystal structure determination reveals a trigonal bipyramidal Au₄Re cluster with the Re atom in equatorial position.If [(Ph₃PAu)₄Re(CO)₄]⁺ is reacted with PPh₄Cl, a cation [Ph₃PAu]⁺ is eliminated as Ph₃PAuCl, and the neutral cluster [(Ph₃PAu)₃Re(CO)₄] (2) is formed.It combines with excess [(Ph₃PAu)₄Re(CO)₄]⁺ to afford the cluster cation, [(Ph₃PAu)₆AuRe₂(CO)₈]⁺. It crystallizes from CH₂Cl₂ as[(Ph₃PAu)₆AuRe₂(CO)₈]PF₆ · 4CH₂Cl₂ (3 · 4CH₂Cl₂). In [(Ph₃PAu)₃Re(CO)₄] the metal atoms are arranged in form of a lozenge while in [(Ph₃PAu)₆AuRe₂(CO)₈]⁺ two Au₄Re trigonal bipyramids are connected by a common axial Au atom.The treatment of [(Ph₃PAu)₄Re(CO)₄]⁺ with KOH and Ph₃PAuCl in methanol yields the cluster cation [(Ph₃PAu)₆Re(CO)₃]⁺, which crystallizes with from CH₂Cl₂ as [(Ph₃PAu)₆Re(CO)₃]PF₆ · CH₂Cl₂ (4 · CH₂Cl₂). The metal atoms in this cluster form a pentagonal bipyramid with the Re atom in the axial position.     

Isolation, X-ray single crystal and theoretical study of quinquevalent metal oxoisopropoxides, Nb6O8(PrO)14(PrOH)2 and Re4O6(OPr)10

Oxoisopropoxide complexes of niobium, Nb₆O₈(ⁱPrO)₁₄(ⁱPrOH)₂ (I), and rhenium, Re₄O₆(OⁱPr)₁₀ (II), were isolated as byproducts of anodic oxidation of these metals in ⁱPrOH in the presence of LiCl as conductive additive. The common feature of both structures consists in the occurrence in their molecules of an M₄O₁₆ planar core formed in the formal absence of M-M bonding for I and in the presence of electron-deficient but surprisingly short (Re-Re 2.52-2.54 Å) metal-metal bonds in II. The stability of this core for the oxoalkoxide derivatives of rhenium(V,VI) and niobium(V) and poor stability for those of tantalum(V) are discussed based on the results of quantum-chemical calculations.     

Reductive thiocyanolysis of tetraoxorhenate (VII): Synthesis, crystal structure, catalytic oxidation and kinetic studies of (PPh4)2[Re(NCS)6] and (PPh4)2[ReO(NCS)5]

Thiocyanate ions reduce perrhenate in aqueous acidic solution, and on addition of a suitable countercation (PPh₄Cl) afford (PPh₄)₂[Re(NCS)₆] (1) and (PPh₄)₂[ReO(NCS)₅] (2), which have been confirmed by X-ray crystallography. The kinetics of the above reaction has been studied. Both the complexes exhibit efficient and highly selective catalytic epoxidation ability in the presence of NaHCO₃ as a co-catalyst and competent catalytic properties in the selective oxidation of alcohols to the corresponding aldehydes or ketones in the presence of pyrazole as an additive and sulfides to sulfoxides and sulfones. H₂O₂ was used as the terminal oxidant in all the above-mentioned oxidation reactions.     

Synthesis and problematic crystal structures of [Al(CH3CN)6][MCl6]3(CH3CN)3 (M=Ta, Nb or Sb)

Compounds of formula [Al(CH₃CN)₆][MCl₆]₃(CH₃CN)₃ (M=Ta (1); Nb (2); Sb (3)) have been synthesized from the reactions of MCl₅ and AlCl₃ in acetonitrile and characterized by X-ray crystallography. Complex 1 crystallizes in the tetragonal space group P4/mbm with a = B = 10.408(2), C = 7.670(3) Å, V = 830.9(4) ų and Z = 2/3. Complex 2 crystallizes in the tetragonal space group P4/mnc with a = B = 330(a), C = 15.320(3) ų V = 1634.8(4) ų and Z = 4/3. Complex 3 also crystallizes in the tetragonal space group P4/mnc with a = B = 10.313(1), C = 15.238(2) Å, V = 1621.0(1) ų and Z = 4/3. The non-integer Z values for complexes 1–3 result unusual problems of disorder and/or twinning in these crystal structures due to their high symmetry. The M---Cl distances range from 2.329(3) Å in the Ta complex to 2.355(1) Å in the Sb complex, while the Al---N distances are similar in all three complexes, ranging from 1.92(1) to 1.97(1) Å, respectively. Complexes 1–3 are the first structurally characterized complexes that contain a (hexaacetonitrile)aluminum(III) cation.     

Production, Purification, and Characterization of Recombinant Human Hemoglobin Rainier Expressed inSaccharomyces cerevisiae

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the β145 tyrosine is substituted with cysteine. The α and β^Rainierglobin cDNAs were cloned in a high copy number vector and expressed inSaccharomyces cerevisiaeunder the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual α and β^Rainierglobin chains. Coexpression of both α and β^RainiercDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the α and β chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A₀. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.     

Spectral properties and bio-activity of copper(II) clofibriates, part III: crystal structure of Cu(clofibriate)2(2-pyridylmethanol)2, Cu(clofibriate)2(4-pyridylmethanol)2(H2O) dihydrate, and Cu2(clofibriate)4(N,N-diethylnicotinamide)2

New copper(II) clofibriates (clof, {2-(4-chlorophenoxy)-2-methylpropionic or 2-(4-chlorophenoxy)isobutyric acid}) of composition Cu(clof)₂L₂ (where L=2-pyridylmethanol (2-pymeth) (1), N-methylnicotinamide (Menia) (4), N,N-diethylnicotinamide (Et₂nia) (5), isonicotinamide (isonia) (7) or methyl-3-pyridylcarbamate (mpc) (8)), [Cu(clof)₂(4-pymeth)₂(H₂O)] · 2H₂O (4-pymeth=4-pyridylmethanol) (2 · 2H₂O) and Cu(clof)₂L (where L=4-pymeth (3) or Et₂nia (6)) have been prepared and spectroscopically characterized. All the Cu(clof)₂L₂ compounds seem to possess distorted octahedral copper(II) stereochemistry with differing tetragonal distortions. An X-ray analysis of 1 was carried out and it featured a tetragonal-bipyramidal geometry around the copper(II) atom. X-ray analysis of 2 · 2H₂O featured a square-pyramidal geometry around copper(II) atom. Both the Cu(clof)₂L compounds seem to consist of a binuclear unit of tetracarboxylate type bridging. An X-ray analysis of 6 revealed typical binuclear paddle-wheel type structure, consisting of two copper(II) atoms in square-pyramidal geometry bridged by four carboxylate anions in the xy-plane. All complexes under study were characterized by EPR and electronic spectroscopy. The antimicrobial effects have been tested on various strains of bacteria, yeasts and filamentous fungi.     

人造血相关PBX相互作用蛋白的原核表达及纯化鉴定

目的:构建带His标签的人造血相关PBX相互作用蛋白(HPIP)的原核表达载体,获得His-HPIP融合蛋白,并对其生物学功能进行初步检测。方法:以本实验室保存的pcDNA3.0-HPIP质粒为模板,采用PCR技术扩增HPIP编码序列,将其插入载体p ET-28a(+)中,经Bam HⅠ和HindⅢ双酶切鉴定后转化大肠杆菌Rossate株进行小量诱导,挑选能诱导出His-HPIP的菌液进行融合蛋白的纯化,采用SDS-PAGE和Western印迹检测融合蛋白的纯化效果,采用GST pull-down技术对蛋白的生物学功能进行初步鉴定。结果:双酶切和测序结果表明His-HPIP原核表达质粒构建成功;His pull-down实验证实His-HPIP蛋白和雌激素受体α存在相互作用,说明生物学活性良好。结论:原核表达并纯化出His-HPIP融合蛋白,为进一步研究HPIP在肿瘤发生发展中的功能奠定了基础。     

大连蛇岛蝮蛇类凝血酶基因在毕赤酵母中的克隆表达及分离纯化(英)

根据同源性分析设计引物,通过RT-PCR方法从大连蛇岛蝮蛇毒腺总RNA中合成扩增出类凝血酶基因,之后将该基因克隆到表达载体pPIC9K中,经电激转化后整合至毕赤酵母细胞基因组中.经筛选得到甲醇快速生长型转化子His⁺Mut⁺在500 ml摇瓶中培养,甲醇诱导分泌表达.上清液中重组类凝血酶是通过两步柱层析得到：Q Sepharose FF和Benzamidine-Sepharose 4BCL.与天然蛇毒类凝血酶一致,分泌表达的重组类凝血酶具有较强的酯酶活性,但精氨酸甲酯如TAME的水解活性较弱.此重组类凝血酶在37℃中性溶液中保存过夜将分解成小肽,但在0℃下很稳定.该酶的最适pH为8.0.     

The preparation, characterisation and low temperature X-ray structure of Ru6(η-μ4-CO)2(CO)13(η6-C6Me6)

The new organometallic cluster (η²-μ₄-CO)₂(CO)₁₃(η⁶-C₆Me₆) has been prepared by the thermolysis of Ru₃(CO)₁₂ with hexamethylbenzene in octane and characterised by a single crystal X-ray diffraction study. It is isostructural with the known cluster Ru₆(η²-μ₄-CO)₂(CO)₁₃(η⁶-C₆H₃Me₃) and the metal core constitutnts the same tetrahedral Ru₄ unit with two edge-bridging Ru atoms. The mesitylene derivative has been shown to undergo rearrangement to afford the octahedral carbido cluster Ru₆C(CO)₁₄(η⁶-C₆H₃Me₃), but this conversion is not observed for the new hexamethylbenzene derivative.     

The amyloidogenic region of the human prion protein contains a high affinity (Met)2(His)2 Cu(I) binding site

The prion protein (PrP^c) is a cuproprotein implicated in a number of human neurodegenerative diseases. Although many physiological functions have been ascribed to PrP, its potential to act as a neuronal antioxidant, based in part on its copper binding ability, is controversial and unresolved. A number of studies have shown that copper bound to PrP^c is not redox silent, and recent data shows that the Cu(II) sites at histidines 96 and 111 display reversible electrochemistry. Reversible electrochemistry implies redox cycling whilst the metal remains bound and with the absence of permanent oxidation or reduction of the protein. Despite this indirect evidence of Cu(I) binding to PrP, the nature of the Cu(I) binding site/s is unclear, although previous extended X-ray absorption fine structure (EXAFS) data has implicated methionines in the Cu(I) binding site. Using spectroscopic techniques we find that the PrP region encompassing histidines 96 and 111 can bind a Cu(I) ion in a site comprising His 96, His 111, Met 109 and Met 112. The four-coordinate (His)₂(Met)₂ Cu(I) site has a K_d = 10⁻¹⁵–10⁻¹² M indicative of high affinity. Mutation of histidine residues reduces the Cu(I) affinity. Although alluding to the fact the PrP could act in a direct superoxide dismutase-like fashion, the Cu(I)–PrP(91–124) site and affinity is comparable to that observed for bacterial periplasmic Cu(I) transporters.     

Spectroscopy and crystal structure of neodymium coordination compound with glycine: Nd2(Gly)6·(ClO4)6·9H2O

Neodymium complex compound with glycine: Nd₂(Gly)₆·(ClO₄₆·9H₂O was synthesized and obtained in the form of monocrystals. Absorption spectra recorded in the region of 8000–35 000 cm^-1 were measured along the crystallographic axes. Intensities of the f-f transitions were analysed on the basis of the Judd theory. The X-ray crystal structure determination of the complex is reported. Crystals are triclinic, space group P$\text{I}$, with a = 11.554(4) Å, b = 14.108(1) Å, c = 15.660(3) Å, α = 97.14(1)°, β = 102.82(2)°, γ = 105.28(1)°, V = 2355.25 ų Z = 2, M.W. = 1495.4, D_c = 2.129)(3) g cm^-3, D^m = 2.103(1) g cm^-3. The structure was solved by Patterson's method and successive Fourier syntheses giving the locations of all nonhydrogen atoms. The final R factor was 0.062 and R_w = 0.073 for 12869 reflections with |F_o| > 5σ|(F_o)|. The asymmetric unit consists of a dimeric formula unit. The coordination polyhedron of Nd atoms comprises seven oxygen atoms from glycine and two from water molecules. The neodymium-glycine bonding mode is compared with that of the calcium-glycine complex.     

Decomposition and superoxide dismutase activity of the copper complex of D-penicillamine (Cu(II)6Cu(I)8 (D-penicillamine)12 Cl)5−

The possible time- and/or light-dependent decomposition of the purple Cu(I), Cu(II)-complex of D-penicillamine (Cu(II)₆Cu(I)₈(D-penicillamine)₁₂Cl)^5? was examined. Superoxide dismutase activity of the freshly prepared complex was assayed using the nitroblue tetrazolium assay. The formazan colour formation was inhibited by 50% in the presence of approximately 500 μM copper. Ageing of the copper complex, especially in the light, resulted in a marked increase of EDTA-sensitive activity. Upon gel chromatography of the aged samples the original low inhibitory activity was restored. All EDTA-sensitive inhibitory activity was found in a clearly separated low M_r copper-containing fraction. Aerobic irradiation with a tungsten lamp at 30 °C accelerated the decomposition of (Cu(II)₆Cu(I)₈(D-penicillamine)₁₂Cl)^5?. ?_Cu518 = 1800 M^?1 cm^?1 dropped to ?_Cu640 = 60 M^?1 cm^?1. The photochemical conversion of (Cu(II)_6? Cu(I)₈(D-penicillamine)₁₂Cl)^5? was complete within 48 h. Due to the identical electronic absorption profile of both, the decomposition product and Cu(II) D-penicillamine disulphide the latter complex was assigned to be the unknown low M_r copper-compound. Circular dichroism and electron paramagnetic resonance measurements support this conclusion.     

Pyruvate Kinase ofTrypanosoma brucei:Overexpression, Purification, and Functional Characterization of Wild-Type and Mutated Enzyme

A procedure was developed for overexpression ofTrypanosoma bruceipyruvate kinase inEscherichia coli.The enzyme was purified to near-homogeneity from the bacterial lysate by first removing nucleic acids and contaminating proteins by protamine sulfate precipitation and subsequent passage over a phosphocellulose column. The purified protein is essentially indistinguishable in its physicochemical and kinetic properties from the enzyme purified from trypanosomes. Furthermore, experiments were undertaken to locate the binding site of the allosteric effector fructose 2,6-bisphosphate. Regulation of pyruvate kinase by this effector is unique to trypanosomes and related protozoan organisms. Therefore, a three-dimensional structure model of the enzyme was made, and a putative effector-binding site could be identified in an interdomain cleft. Four residues in this cleft were mutated, and the mutant proteins were produced and purified, using the same methodology as for the wild-type pyruvate kinase. Some mutants showed only minor changes in the activation by the effector. However, substitution of Arg22 by Gly resulted in a 9.2-fold higherS_0.5for phosphoenolpyruvate and a significantly smallerk_catthan the wild-type enzyme. Furthermore, the apparent affinity of this mutant for the allosteric effectors fructose 1,6-bisphosphate and fructose 2,6-bisphosphate was 8.2- and 5.2-fold lower than that of its wild-type counterpart. Effector binding was also affected, although to a lesser extent, in a mutant Phe463Val. These data indicate that particularly residue Arg22, but also Phe463, are somehow involved in the binding of the allosteric effectors.     

Supramolecular assemblies of new 2-D complexes: Syntheses, crystal structures, spectroscopic and magnetic properties of {[MnAu2(CN)4(NITpPy)2(H2O)2]}n and {[Co(N(CN)2)2(NITpPy)2(H2O)2]}n

Two new complexes, {[MnAu₂(CN)₄(NITpPy)₂(H₂O)₂]}_n (1) and {[Co(N(CN)₂)₂(NITpPy)₂(H₂O)₂]}_n (2), have been synthesized and characterized. The single-crystal X-ray analysis for the complexes 1 and 2 demonstrates that each M(II) (M = Mn or Co) ion assumes a distorted octahedral MN₄O₂ coordination polyhedron. Four nitrogen atoms come from the cyanide groups and the pyridyl rings in a common plane, and two oxygen atoms come from the H₂O molecules in trans-positions. The structures of complexes 1 and 2 illustrate that aurophilicity and/or hydrogen bonding interactions play important roles in increasing dimensionality. Magnetic investigations on complexes 1 and 2 show the presence of weak antiferromagnetic interactions.     

Synthesis and optical study of Ln(III)(tetramethylurea)(AsF6)3 (Ln = LaLu,Y) and crystal structure for Ln = Eu [Eu(TMU)6(AsF6)3]

The fluorescence spectrum of Eu³⁺ recorded at a temperature of 12 K between 14.000 and 20.000 cm^?1 shows transitions from the excited stare ⁵D_o to the Stark components of the lowest ⁷F_j. The optical analysis suggests an octahedral site symmetry for the rare earth ion which is confirmed by the three-dimensional crystal determination. The highly disordered crystal structure refined in space group F23 to an R-factor of 13.2%. Both the europium and the arsenic ions are located in special positions of point symmetry 23(T). The EuO bond distance is 2.28 Å. The value of the B_q^k parameters was determined.