质谱法分析大肠癌组织中踝蛋白的磷酸化修饰

踝蛋白的磷酸化修饰,特别是肿瘤等病理条件下的踝蛋白磷酸化状态,与肿瘤的发病、转移机理密切相关。本研究采用盐析、离子交换层析和电泳分离并纯化了人大肠癌组织中的踝蛋白(Talin),经胰酶水解获得其肽段混合物,进一步分别利用固定化Fe3+亲和层析和TiO2亲和层析在酸性条件下对其中磷酸化修饰肽段进行吸附,并以1%氨水进行洗脱。在Michrom Magic C18色谱柱上,以A:99%水+1%乙腈+0.1%甲酸和B:99%乙腈+1%水+0.1%甲酸两种流动相进行梯度洗脱分离,采用ESI质谱进行依赖数据的二级子离子扫描。结果显示,固定化Fe3+富集到8个磷酸化肽段而TiO2富集到9个磷酸化肽段。本研究提供了一种快速、准确地鉴定从人大肠癌组织中分离表征踝蛋白的方法。     

Rapid method to isolate serum amyloid P component from human plasma. Characterization of the isolated protein.

A rapid and reproducible method to isolate serum amyloid P component from healthy human plasma has been developed. It uses affinity chromatography on an agarose column followed by anion-exchange chromatography. It was found that the isolated compound has a significantly different isoelectric point (pI 5.7) from that reported previously (pI 4.1). The new data are in good agreement with calculated values determined from the amino acid composition of the protein.     

Identification of yeast oxidized proteins: chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast

The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase liquid chromatography (RPLC) on a C(8) column. Oxidized protein fractions from RPLC were then trypsin digested and the peptide cleavage fragments identified by tandem mass spectrometry (MS/MS). Slightly over 400 proteins were identified. Sites of carbonyl formation were found in roughly one fourth of these proteins. Oxidation on other amino acids in carbonylated peptides was seen in 32 cases while carbonylation was absent in 96 of the oxidized proteins observed. Although there are large numbers of potential oxidation sites, oxidation seemed to be restricted to a small area in most of the proteins identified. Sometimes multiple amino acids in the same tryptic peptide were oxidized. A second trend was that more than 8% of the proteins identified appeared in more than one of the RPLC fractions. Based on the position of the peptides identified in the primary structure of protein candidates derived from databases it was concluded that this occurred by fragmentation of a parent protein. It is not clear from the data whether the fragmentation process was of enzymatic or oxidative origin. Finally, peptides from two or more proteins occurred together in more than one reversed phase fraction with 2% of the proteins identified. This data was interpreted to mean that this was the result of protein cross-linking.     

Characterization of calcium and phospholipid dependent protein kinase in isolated rat adipocytes

Calcium and phospholipid-dependent protein kinase (protein kinase C) from isolated rat adipocytes has been partially purified using DEAE-Sepharose CL-6B and characterized. The enzyme was shown to have similar properties as the kinase isolated from brain or spleen. When histone was used as substrate, an equal amount of cAMP-dependent and calcium and phospholipid-dependent kinase activity was detected from the DEAE Sepharose CL-6B fractions. The major part of protein kinase C (72%) was isolated from the soluble adipocyte fraction. Of the membranous fractions, the plasma membrane exhibited the highest specific activity. The protein kinase preparations bound [3H]-phorbol-12,13-dibutyrate (PDBU) with high affinity (Kd = 2 nM) and the number of PDBU binding sites per cell was calculated to 63 000.     

Self-cleavable stimulus responsive tags for protein purification without chromatography

A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle.     

Adsorption of a phospholipid-hydroperoxide glutathione peroxidase into phospholipid monolayers at the air-water interface

The interfacial behavior differences of two glutathione peroxidase isoforms have been investigated. The first isoform is the phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12) (GPx-4) isolated from rat testes and the second one is the cytosolic glutathione peroxidase (EC 1.11.1.9) (GPx-1) from bovine erythrocytes. Injected in the subphase buffer of a Langmuir trough, GPx-4 was able to adsorb quickly at the air-water interface whereas the GPx-1 was not. Then, the protein interaction with phospholipid monolayers was explored. Indeed, a monolayer of phospholipids containing a different number of polyunsaturated fatty acyl chains was prepared at the air-water interface. Under each kind of monolayer, the protein solution was injected and its adsorption was visualized by the measurement of successive pressure-area isotherms. We have, then, determined the molecular area increase due to the protein adsorption. It was found that the GPx-4 is adsorbed in each kind of monolayer tested whereas no molecular area increase was detected with the GPx-1. This indicates that the GPx-4 has a higher affinity for the interface, recovered or not by lipids, than the GPx-1. Moreover, the GPx-4 presents a different affinity for the phospholipid monolayers depending on the number of polyunsaturated fatty acyl chains.     

An immunoaffinity column for the determination of peanut protein in chocolate.

An immunoaffinity column was prepared from rabbit polyclonal antiserum for the determination of peanut protein from food matrixes. The anti-peanut immunoglobulin G was isolated from antiserum by affinity chromatography on a column coupled with peanut protein and then attached to an AminoLink gel. The column was applied to the determination of peanut protein in chocolate after extraction, immunoaffinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Overall recoveries from chocolate spiked with 0.2-3.2 micrograms/g of peanut protein averaged 77% (range, 72-84%), and the minimum detection limit was 0.1 microgram/g. Chromatography of extracts with the column improved detection limit and eliminated the matrix effect experienced with direct ELISA of chocolate extracts.     

Lactoferrin from human breast milk and from neutrophil granulocytes. Comparative studies of isolation, quantitation, characterization and iron binding properties

The isolation and properties of lactoferrin from human breast milk and from neutrophilic granulocytes were investigated. Human breast milk lactoferrin was purified by means of heparin-sepharose or Cibacron Blue affinity chromatography. Quantitative recovery using these two methods was comparable but Cibacron Blue affinity chromatography allowed for isolation of a more homogenous protein. Lactoferrin could only be isolated from human neutrophilic granulocytes by sequential use of antibody affinity followed by non-specific affinity chromatography. Both breast milk lactoferrin and granulocyte lactoferrin were separated into apo and iron-rich species by SDS polyacrylamide gel chromatography. Iron binding is accompanied by a conformational change in tertiary structure associated with more rapid electrophoretic migration. The isoelectric point of both human breast milk lactoferrin and human granulocyte lactoferrin is 5.5-6.2. Both types of lactoferrin have similar iron binding properties with release of iron from the one binding site occurring at pH 5.2-6.0 while the other binding site holds on to iron down to pH 3.6-3.2. Despite the high affinity for iron the percentage saturation of native lactoferrin is low, that for breast milk lactoferrin averaging 12-25% and that for granulocyte lactoferrin less than 10%.     

Chromatographic and electrophoretic studies of circulating immune complexes in plasma

The protein nature of soluble immune complexes from fresh plasma was studied by combining several analytical biochemical techniques. Free immunoglobulins (Ig) G were separated from larger immune complexes by gel permeation chromatography. In a second step, immune complexes, free IgA and IgM were isolated by protein-A and protein-G affinity chromatography and analysed by two-dimensional gel electrophoresis. Sixteen plasma samples from healthy donors were analysed and evaluated visually. Their protein profiles on the gels turned out to be similar, showing only slight quantitative differences. In one case, additional proteins were detected. To prove the ability of the method, immune complexes were analysed from four plasma samples that showed macro creatine kinase type 1, a complex formation between creatine kinase BB and IgG. This methodology can be used for the examination of immune complexes of unknown protein composition in serum or plasma.     

Identification of the binding of sceptrin to MreB via a bidirectional affinity protocol

A bidirectional affinity system was used to screen for marine natural products that bound to Escherichia coli proteins. A system was developed and applied to isolate the natural product sceptrin from an Agelas conifera extract and its affinity partner MreB from E. coli lysate. The use of a dual immunoaffinity fluorescent (IAF) tag permitted this process to co-immunoprecipitate the bacterial equivalent of actin, MreB, from E. coli lysate. MreB was subsequently validated as a target for sceptrin using a resistance mapping approach. The combination of these studies suggests that natural products and their protein targets can be isolated in concert using a melody of forward and reverse affinity matrices. While the structure of sceptrin was elucidated by NMR analysis, the bulk of effort was conducted without knowing the structure of the natural product, thereby elevating a key bottleneck in the development of high-throughput methods for natural product discovery.     

Biosynthesis of isoprenoids. purification and properties of IspG protein from Escherichia coli

[Structure: see text]. The IspG protein is known to catalyze the transformation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the nonmevalonate pathway of isoprenoid biosynthesis. We have found that the apparent IspG activity in the cell extracts of recombinant Escherichia coli cells as observed by a radiochemical assay can be enhanced severalfold by coexpression of the isc operon which is involved in the assembly of iron-sulfur clusters. The recombinant protein was isolated by affinity chromatography under anaerobic conditions. With a mixture of flavodoxin, flavodoxin reductase, and NADPH as the reducing agent, stringent assay methods based on photometry or on 13C NMR detection of multiply 13C-labeled substrate/product ratios afforded catalytic activities greater than 60 nmol mg(-1) min(-1) for the protein "as isolated" (i.e., without reconstitution of any kind). Lower apparent activities were found using photoreduced deazaflavin as an artifactual electron donor, whereas dithionite was unable to serve as an artificial electron donor. The apparent Michaelis constant for 2-C-methyl-D-erythritol 2,4-cyclodiphosphate was 700 microM. The enzyme was inactivated by EDTA and could be reactivated by Mn2+. The pH optimum was at 9.0. The protein contained 2.4 iron ions and 4.4 sulfide ions per subunit. The replacement of any of the three conserved cysteine residues afforded mutant proteins which were devoid of catalytic activity and contained less than 6% of Fe2+ and less than 23% of S2- as compared to the wild-type protein. Sequence comparison indicates that putative IspG proteins of plants, the apicomplexan protozoan Plasmodium falciparum, and bacteria from the Bacteroidetes/Chlorobi group contain an insert of about 170-320 amino acid residues as compared with eubacterial enzymes.     

Intact cell matrix-assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression

Here we demonstrate for the first time the application of intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione-S-transferase (GST) gene under an arabinose-inducible promoter as a model system. Using ICM-MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose-induced condition. Furthermore, recombinant GST protein was purified by a single-step affinity purification using a glutathione Sepharose 4B affinity column from arabinose-induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose-induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose-mediated regulation of the ara-operon was followed using the ICM-MS technique. All the results obtained from ICM-MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases.     

Properties of (Mg2 + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: influence of Mg2+, Ca2+, ATP, and protein activator.

Erythrocyte membranes prepared by three different procedures showed (Mg2+ + Ca2+)-ATPase activities differing in specific activity and in affinity for Ca2+. The (Mg2+ + Ca2+)-ATPase activity of the three preparations was stimulated to different extents by a Ca2+-dependent protein activator isolated from hemolysates. The Ca2+ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2-200 microM or lowering the Mg:ATP ratio to less than one shifted the (Mg2+ + Ca2+)-ATPase activity in stepwise hemolysis membranes from mixed "high" and "low" affinity to a single high Ca2+ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strength, or membranes prepared by the EDTA (1-10 mM) procedure, did not undergo the shift in the Ca2+ affinity with changes in ATP and MgCl2 concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg2+ + Ca2+)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.     

Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography

A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.     

Extracellular oxidases purified fromCoriolus versicolor

Studies on the extracellular enzymes ofCoriolus versicolor have resulted in the isolation and purification of several proteins that have the potential to act as redox enzymes.C. versicolor was cultured on a glucose-amino acid medium in a large-scale fermenter (60 L) with 2,5-xylidine added to induce the production of extracellular laccase. Proteins were precipitated from the growth medium with ammonium sulfate, and separated by ion-exchange chromatography on DEAE-Sephadex. Further separation of glycoproteins was achieved by affinity chromatography on Concanavalin-A-Sepharose. Polyacrylamide gel electrophoresis on SDS (sodium dodecyl sulfate) and LDS (lithium dodecyl sulfate) gels, isoelectric focusing, and chromatofocusing have been used to establish purity of the proteins and their isoelectric points. Laccase B has been isolated and separated into five fractions by chromatofocusing, with isoelectric points of the fractions varying between pH 4.5 and 6.5. The relative specificity of these fractions towards monophenolic and diphenolic substrates has been investigated. Laccase A was found to differ from laccase B in showing only two bands on isoelectric focusing, with isoelectric points between pH 3.0 and 3.5. Two other proteins isolated from the growth medium were both hemecontaining proteins with interesting spectral properties. One was a “peroxidasetype” heme that could bind carbon monoxide to the iron in the heme, suggesting that the heme may bind oxygen and so function as an oxidase. It reacted with hydrogen peroxide to liberate hydroxyl radicals, but this reaction with hydrogen peroxide resulted in the destruction of the heme center. The real role of this protein is unclear, but several possibilities will be investigated. The second heme-containing protein isolated had different spectral properties from the “peroxidase-type” heme previously described. It had spectral characteristics of a b-type cytochrome in association with a flavin prosthetic group. It appeared to have some similarities to cellobiose oxidase, a heme flavoprotein previously isolated fromSporotrichum pulverulentum, although its molecular weight was 50,100 daltons compared with the 93,000 reported for cellobiose oxidase. Further characterization of this protein will be described.     

Affinity sorbent from abscisic acid — Agarose A-6

Optimal conditions are found for synthesizing an affinity sorbent from abscisic acid (ABA) and aminopropylagarose A-6. A protein with high specific affinity for ABA is isolated from the microsomal fraction of four-day cotton sprouts using the synthesized sorbent.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (99871) 162 70 71. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 157–159, March–April, 2000.     

牛天然纤溶酶原环状结构域K1—3的获得

采用Ｌｙｓ－Ｓｅｐｈｅｒｏｓｅ４Ｂ亲和层析,从新生牛血清中分离提纯的牛纤溶酶原（ｐａｌｓｍｉｎｏｇｅｎ,ｐｇｎ）。利用ＳＤＳ－聚丙烯酰胺凝胶电泳分析,对其纯度和相对分子质量进行了鉴定。利用猪胰弹性蛋白酶酶切纤溶酶原获得了环状结构域Ｋ１－３,采用Ｌｙｓ－Ｓｅｐｈｅｒｏｓｅ４Ｂ亲和层析对该片段进行了分离提取。     

Affinity Chromatography of Mushroom Tyrosinase

Abstract

The purification by column chromatography of a phenol-oxidizing enzyme, mushroom tyrosinase, was investigated using solid phase adsorbents designed to have specific affinity for the enzyme. Sepharose 4B, aminophenyl-bearing porous glass, and p-aminobenzylcellulose were chemically modified to introduce phenolic, catecholic, or benzoic groups on the polymer surface. The resulting preparations were tested for their effectiveness in separating tyrosinase from an impure protein mixture. The phenolic and benzoic polymers displayed no specific affinity for tyrosinase. Aminophenyl glass, with or without an attached phenolic group, adsorbed appreciable quantities of protein nonspecif-ically, thus complicating studies of its tyrosinase affinity properties. Dopamine, a dihydroxyphenyl derivative, was bound to Sepharose and was found to be effective in retaining tyrosinase at pH 5.5; elution of the enzyme by washing at pH 8.8 resulted in its purification by a factor of 10 to 14. Enzymatic oxidation of the adsorbent limited the number of purification cycles which could be carried out on a single column.     

Isolation and purification of cat albumin from cat serum by copper ion affinity chromatography: further analysis of its primary structure

Proteins, regardless of their origin, have to be highly purified, particularly from the immunochemical point of view, if they are to be used to study their allergenicity. It is shown that cat albumin, a highly potent allergen for cat-sensitive humans, can be isolated and purified from cat serum using immobilized metal ion affinity chromatography (copper ions) instead of a salting-out process or precipitation with alcohol, techniques generally used for the preparation of serum proteins. During the process described, immunoglobulins are concomitantly isolated in a relatively pure form. Cat albumin amino acid composition and sequence were analysed after an ultimate purification by ion-exchange chromatography. The highest homology (greater than 80%) was found with the rat serum albumin.