重组葡激酶的分离纯化

在构建出高表达,高活性的葡激酶工程菌株的基础上,对表达产物进行了分离纯化研究。经离子交换纯化后,纯度达８５％－９０％,回收率为５０％－６０％,经凝胶过滤后,纯度达９９％以上,回收率为６０％,经ＳＤＳ－ＰＡＧＥ测定,相对分子质量为１５．５＊１０＾３,比活为１．０＊１０＾５,Ｎ端氨基酸序列与献报道的相符。     

Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases

Abstract

Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.     

葡激酶及其化学修饰研究进展

葡激酶(SAK)是一种由金黄色葡萄球菌分泌的含有136个氨基酸残基的蛋白质,大量研究证明它具有潜在的溶血栓特性。但葡激酶是一种外源蛋白,注入体内会引起不良反应,解决这些问题的重要途径就是对蛋白质进行化学修饰。本综述SAK的结构、洛栓机理、化学修饰及临床研究等。     

重组葡激酶工程菌的构建方法

目的：利用N端缺失10个氨基酸的葡激酶（recombinant staphylokinase,rSaK）重组质粒,构建了表达可溶性rSaK126蛋白的工程菌,并研究不同条件下工程菌诱导表达目的蛋白含量的差异及纯化途径。 方法：采用细菌活化和培养方法诱导目的蛋白,并用SDS-PAGE测其含量,应用层析技术纯化蛋白。 结果：成功构建表达重组葡激酶的工程菌,表达的重组葡激酶蛋白约占菌体总蛋白的50％,经纯化后回收率为60%,纯度达99%以上。结论：成功构建高效表达重组葡激酶的工程菌,并获得了高含量、高纯度的目的蛋白。     

Crystallization and preliminary X-ray investigation of the recombinant Trypanosoma brucei rhodesiense calmodulin

Bipyramidal crystals of the recombinant calmodulin from Trypanosoma brucei rhodesiense were obtained by vapor diffusion against 55% (v/v) 2-methyl-2,4-pentanediol in 0.05 M cacodylate buffer, pH 5.6. When few nucleation events occurred, crystals grew to 0.25 × 0.25 × 1.20 mm. The space group of the crystal is I4₁22, with unit cell dimensions a = b = 56.88 Å, c = 230.11 Å, α = β = γ = 90°, z = 16. The molecular mass and volume of the unit cell suggest that there is one molecule in the asymmetric unit. The I/σ(I) ratio for data at 3.0 Å resolution was 3.67, indicating that the final structure can be refined at higher resolution. Molecular replacement methods and the PC-refinement technique have not yet yielded the structure under a variety of search conditions. We are currently investigating the multiple isomorphous replacement approach to determine this crystal structure. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic studies on Type III antifreeze protein.

Type III antifreeze protein, more specifically the recombinant QAE-Sephadex-binding isoform, has been crystallized in 50-55% saturated ammonium sulfate, 0.1 M sodium acetate, pH 4.0-4.5. The resultant crystals belong to the orthorhombic space group P212121 with a = 32.60 A, b = 39.00 A, and c = 46.57 A and diffract to at least 1.7 A. A set of 1.7-A native data has been collected, with completeness 93.4% and Rsym of 0.069. Initial screening for heavy-atom derivatives has yielded a Pt-bound derivative.     

重组葡激酶的基因克隆与序列测定

从烧伤病房病人烧伤感染创面分离到 9株金黄色葡萄球菌 ,经纤溶活性测定 ,选纤溶活性最高的作为 PCR扩增模板。将扩增出的葡激酶基因片段插入 p UC1 8载体质粒 ,转化大肠杆菌 DH5 α,筛选出 PUC- SAK阳性克隆 ,经 DNA序列测定证实该基因片段为一种新的 DNA序列     

Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.     

Crystallization and preliminary X-ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides.

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 A. Based on the assumption of one 32-kDa monomer per asymmetric unit, the Vm value is 2.83 A3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 A resolution.     

Crystallization and preliminary X-ray diffraction analysis of recombinant pentalenene synthase.

Recombinant pentalenene synthase, a 42.5-kDa sesquiterpene cyclase originally isolated from Streptomyces UC5319 and cloned in Escherichia coli, has been crystallized in space group P6(3) with unit cell dimensions a = b = 183.5 A and c = 56.5 A. Hexagonal prismatic crystals, approximately 0.2 x 0.2 x 0.3 mm, diffract to approximately 2.9 A resolution using monochromatic synchrotron radiation. From the universal (and achiral) building block, farnesyl pyrophosphate, pentalenene synthase catalyzes the formation of four stereocenters in the construction of the three fused five-membered rings of pentalenene; this novel sesquiterpene is a precursor to the pentalenolactone family of antibiotics.     

Crystallization and preliminary X-ray diffraction studies of a bacterial flavohemoglobin protein

A flavohemoglobin protein (FHP) was isolated from Alcaligenes eutrophus and has been crystallized by vapor diffusion methods using PEG 3350 as precipitant. The crystals of the FAD- and heme-containing protein belong to the monoclinic space group P2₁ with unit cell parameters of 52.2 Å, 85.8 Å, 103.9 Å, and 81.8° corresponding to two molecules per asymmetric unit. The crystals diffract at least to a resolution of 2.0 Å and are suitable for an X-ray structure analysis. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray investigation of a recombinant form of rat catechol O-methyltransferase.

Rat catechol O-methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and soluble recombinant catechol O-methyltransferase was produced in bacteria and purified to electrophoretic homogeneity by chromatographic procedures. The purified enzyme has been crystallized by the method of vapor diffusion using polyethylene glycol as precipitant. The space group is P3(1)21 or P3(2)21 with a = b = 51.3 A and c = 168.5 A and one molecule in the asymmetric unit. The crystals diffract beyond 3.2 A and are suitable for three-dimensional X-ray structure determination.     

Crystallization and preliminary crystallographic analysis of tetrahydrodipicolinate-N-succinyltransferase

Crystals of tetrahydrodipicolinate-N-succinyltransferase have been obtained from solutions containing 2-propanol and polyethylene glycol 4,000. These crystals belong to the monoclinic space group P2₁, diffract X-rays to a resolution of 2.2 Å, and contain one trimer per asymmetric unit. © 1996 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic data for class I deoxyribose-5-phosphate aldolase from Escherichia coli: An Application of Reverse Screening

X-ray quality crystals of class I deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate. The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2₁2₁2₁ with cell dimensions a = 183.1 Å, b = 61.4 Å, c = 49.3 Å and a = 179.2 Å, b = 60.5, Å, c = 49.1 Å, respectively. Two molecules in the asymmetric unit are related by a noncrystallo-graphic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 Å. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions. © 1995 Wiley-Liss, Inc.     

重组葡激酶基因在大肠杆菌内的高水平表达及生物学活性研究

将测序后的葡激酶重组质粒ＰＵＣ－ＳＡＫ经酶切后,组装于表达载体ｐＢＶ２２０,构建成ｐＢＶ－ＳＡＫ表达质粒,转化大肠杆菌。重组葡激酶表达水平达６０％～７０％,相对分子质量为 １５ ５００,主要以可溶状态存在于细胞中。生物活性测定证实,重组葡激酶具有很强的纤溶活性。     

Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase.

The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms.     

Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase.

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme. MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P4(1)2(1)2 with a = b = 49.6 A, c = 263.2 A, and alpha = beta = gamma = 90 degrees at -160 degrees C and diffract to at least 2.5 A. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.     

Crystallization and Preliminary X-Ray Diffraction Analysis of Group 5 Mite Allergen fromDermatophagoides pteronyssinus

Crystals of the 14-kDa group 5 allergen fromDermatophagoides pteronyssinus(Der p 5) have been obtained at low pH and diffract to 3-Å resolution using a conventional x-ray source. The crystals belong to tetragonal space group P4₁2₁2 or P4₃2₁2, with unit cell parametersa=b= 114 Å andc= 234 Å. A self-rotation search revealed a 432 point symmetry and thus suggested 96 molecules in one unit cell, hence 12 monomers in each asymmetric unit.     

Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained.     

Crystallization and preliminary X-ray diffraction analysis of bovine seminal plasma PDC-109, a protein composed of two fibronectin type II domains

PDC-109 is a 13 kDa glycoprotein and the major phosphorylcholine- and heparin-binding protein of bull seminal plasma. It is built by an acidic 23-residue N-terminal sequence followed by a tandem of fibronectin type II domains. Full-length PDC-109 was crystallized in complex with o-phosphorylcholine by vapor diffusion in sitting drops. Crystals grew to maximal size of 0.5 × 0.3 × 0.2 mm³, diffract x-rays beyond 2.6 Å resolution, and belong to space group P321 with unit cell dimensions a = b = 93.6 Å, c = 52.7 Å. Proteins 28:454–456, 1997. © 1997 Wiley-Liss, Inc.