Purification of isocitrate lyase from Escherichia coli and watermelon using fast protein liquid chromatography

The enzyme isocitrate lyase has been purified to gel electrophoretic homogeneity from Escherichia coli and watermelon. From cotyledons of the latter source, the enzyme is obtained in less than 8 hours after precipitation with (NH4)2 SO4 followed by fractionation on cationic Mono S microbeads and anionic Mono Q microbeads using Fast Protein Liquid Chromatography (FPLC). From a genetically engineered E. coli strain, in which high-level expression of isocitrate lyase occurs, the enzyme has been purified in one step from the high-speed supernatant using a Mono Q column with FPLC. These purifications, both of which give satisfactory yields, potentiate rapid access to isocitrate lyase from both prokaryotic and eukaryotic sources.     

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

Purification of a high molecular weight membrane protein by fast protein liquid chromatography: the ATPase complex of a thermophilic cyanobacterium

Fast protein liquid chromatography (FPLC) with a strong anion-exchange (Mono Q) column is applied to the purification of a high molecular weight membrane protein. The ATPase complex of the thermophilic cyanobacterium Synechococcus 6716, partially purified by ammonium sulfate precipitation, was fractionated in the presence of the detergent octylglucoside. The ATPase complex containing fractions were eluted by a linear NaCl gradient at about 0.4 M and within 10 min. The FPLC fractions were analyzed for protein and pigment contents and by polypeptide composition. The purest fraction is essentially free of pigments and has a high specific ATP hydrolysis activity (about 1.6 mumol ATP X min-1 X mg protein-1) which is sensitive to N,N'-dicyclohexylcarbodiimide.     

A rapid method for purification of homogenous Legionella pneumophila cytotoxic protease using fast protein liquid chromatography

A purification method was developed to isolate Legionella pneumophila cytotoxic protease in a form suitable for biological assays. Culture supernatant of a clinical isolate of L. pneumophila, Knoxville 1 strain, was used as the starting material. The protease was purified by FPLC on a Mono Q column followed by ultrafiltration. The isolated proteolytic enzyme has a specific activity of 90 azocasein units/mg protein and is a 42 kDa monomeric protein as determined by SDS-PAGE and gel filtration chromatography. It is heat-labile and toxic to a variety of cells e.g. McCoy, SIRC, HeLa, and rhabdomyosarcoma cells, baby hamster and green monkey kidney cells, and human embryonic lung fibroblasts.     

Properties and developmental regulation of the protein phosphatases in Dictyostelium discoideum

The properties and developmental regulation of the protein phosphatases of Dictyostelium discoideum were examined. When crude extracts from vegetative cells were separated on a Mono Q column (FPLC) three protein phosphatase peaks, designated P1, P2 and P3 were found. When aggregation and culmination cells were examined only one protein phosphatase peak was observed. This corresponded to phosphatase P1 of vegetative cells. All three of the vegetative cell phosphatase were inhibited by heparin and mammalian phosphatase inhibitor-2, both of which are specific for type-1 protein phosphatases. Trifluoperazine, which inhibits type-2 protein phosphatases, had little effect on any peaks while levamisole, an alkaline phosphatase inhibitor, stimulated P2, slightly inhibited P3 and had no effect on P1. These results demonstrate the existence of two vegetative phase specific protein phosphatases in D. discoideum and one which occurs during all phases of the life cycle. The protein phosphatases isolated from vegetative cells all appear to be type-1 enzymes.     

A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization

We have recently described a luminal guanosine diphosphatase activity in Golgi-like vesicles of Saccharomyces cerevisiae (Abeijon, C., Orlean, P., Robbins, P. W., and Hirschberg, C. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6935-6939). The presumed in vivo role of this enzyme is to convert GDP into GMP. GDP is a reaction product following outer-chain mannosylation of luminal proteins and a known inhibitor of mannosyltransferases. It is hypothesized that GMP then returns to the cytosol. We have purified this enzyme to apparent homogeneity. Following solubilization from a membrane pellet using a buffer containing Triton X-100, the enzyme was purified on a concanavalin A-Sepharose column followed by Mono Q fast protein liquid chromatography (FPLC) and Superose-12 FPLC columns. After treatment with endoglycosidase H, the deglycosylated active enzyme was applied to a second Mono Q FPLC column and a phenyl-Superose FPLC column. The final enzyme activity was enriched 6500-fold over that of the Triton X-100 extract. The apparant molecular mass of the deglycosylated enzyme is 47 kDa. The purified enzyme is highly specific for guanosine diphosphate, requires Ca2+ for maximal activity, and has a broad pH optimum between 7.4 and 8.2. The apparent Km for GDP is 0.1 mM; the Vmax is 4.9 mmol/min/mg of protein. An enzyme activity with similar substrate specificity has also been detected in membranes of Schizosaccharomyces pombe.     

利用FPLC提纯抗苯丙氨酸羟化酶抗体的Fab及其轻链和重链的分离

 IgGPH_7经木瓜蛋白酶消解产生Fab和Fc,在FPLC仪上,分别用MonoQ,Superose_(12),MonoS柱提纯Fab。提纯的Fab经还原和烷基化后,再用琥珀酸酐修饰,然后用Mono Q柱分离得到轻链和重链。最后通过SDS凝胶电泳和N端顺序测定,以鉴定它的纯度和轻、重链。     

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.     

Establishment of rat-mouse T cell hybridomas that constitutively produce a soluble factor that is needed for the generation of cytotoxic cells: biochemical and functional characterization

Spleen cells from Lewis rats were cultured with 4 micrograms/ml Con A. These cells were then fused with BW 5147 mouse T lymphoma cells. Two hybrid clones (6B2-B8 and 6B2-E6) obtained by fusion formed CGF effectively. It was found that hybrid cells can be boosted to produce higher levels of CGF upon stimulation with Con A. 6B2-B8 express rat T cell markers. CGF formed by 6B2-B8 had a m.w. of 23,000 and 40,000. CGF was eluted from a Mono Q anion-exchange column with an FPLC system at 0.4 to 0.6 M NaCl as a major peak and at 0.8 M NaCl as a minor peak. CGF was eluted as three peaks with pH 4.1, 4.8, and 5.2 from a Mono P chromatofocusing column. CGF from 6B2-B8 does not contain IL-1, IL-2, IL-3, or CSF.     

Partial purification and characterization of masking protein for beta-type transforming growth factor from rat platelets

beta-Transforming growth factor (TGF-beta) is stored in platelets and secreted as a high molecular weight latent form associated with a carrier protein of about 440 KD. This carrier protein could be separated from TGF-beta in 1 N acetic acid and could again mask the activity of TGF-beta under neutral conditions. Therefore, it was named the masking protein of TGF-beta. The masking protein was separated from TGF-beta by gel filtration on a Sephacryl S-300 column or by anion-exchanger FPLC on a Mono Q column in the presence of 6 M urea. Partially purified masking protein from rat platelets neutralized the activity of TGF-beta dose-dependently and was effective at 0.3 microgram/ml. This masking protein could also mask the activity of human TGF-beta, suggesting that it was not species specific. The masking protein was a heat- and acid-stable protein, but was inactivated by treatment with dithiothreitol. The Physiological role of the masking protein in the mechanisms of wound healing and liver regeneration is discussed.     

拮抗菌TG26的鉴定及其抗菌蛋白BI的纯化和部分特性

从丝瓜根部分离出来的桔抗菌ＴＧ２６,根据形态特征和生理生化特性,鉴定为枯草芽抱杆菌（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）。该菌株能分泌大量的抗菌蛋白,经ＳｅｐｈａｄｅｘＧ－１５０柱和ＦＰＬＣＭｏｎｏＱ柱层析后得到单一组分的抗菌蛋白,命名为ＢＩ。ＢＩ能抑制多种植物病原菌的生长,对热稳定,对蛋白酶部分敏感。经ＳＤＳ－ＰＡＧＥ和等电聚焦电泳测定其分子量约１４．５ｋＤ,等电点为５．５８。氨基酸组成分析表明,该蛋白宫含谷氨酸、酪氨酸和脯氨酸;并测定了Ｎ末端氨基酸的部分序列。     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.     

A new retinoid-binding protein with a low molecular weight

Cellular retinoid-binding proteins and nuclear receptors may mediate the intracellular transport and the action of retinoids in the control of differentiation and tumorigenesis. We report a new retinoid-binding protein (Ret BP) with a molecular size of 4,000 that binds retinol, retinoic acid, and some of their derivatives. Purification of Ret BP from chick skin cytosol involved DEAE-Sephadex, Sephadex G-100, and Mono Q column chromatography. The Ret BP-retinoid complex eluted at 195 mM NaCl during Mono Q column chromatography using a 0-300 nM NaCl gradient. Superose-12 column chromatography indicated a molecular size of 4,000 for Ret BP. The binding protein showed a pI of 6.8 on electrofocusing in ampholines of pH 3-10. Ret BP may act as an affinant for retinoids in the cell, and may serve to dispense the ligands to their respective functionally active sites.     

Purification and characterization of a growth factor-like which increases capillary permeability from Vipera lebetina venom

We have investigated the effect of Vipera lebetina venom on capillary permeability and isolated an increasing capillary permeability protein (ICPP) which is devoid of arginine ester hydrolase and phospholipase A2 activities. This protein was purified with a yield of about 0.2% by fast protein liquid chromatography (FPLC) using successively Superose 12, Mono Q, and Mono S columns and by high-pressure liquid chromatography (HPLC) on a C8 reverse-phase column. The purified protein migrated on SDS-PAGE as a band of about 27 kDa under nonreducing conditions and as a band of about 16 kDa under reducing conditions. Chromatography on a C8 column of reduced and alkylated protein yielded a single peak suggesting that this protein is homodimeric. This protein was refractory to Edman degradation chemistry. We used successfully a chemical unblocking involving the incubation of the protein with HCl in anhydrous methanol. The N-terminal amino acid sequence clearly shows considerable similarity to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).     

Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.     

Purification of plasmid DNA by fast protein liquid chromatography on superose 6 preparative grade

We were able to reduce both the time and the use of hazardous chemicals associated with the previous plasmid isolation methods of high-pressure liquid chromatography and CsCl gradient centrifugation by employing fast protein liquid chromatography (FPLC). Plasmid was first crudely prepared from bacterial cultures by a standard alkaline lysis method. After an alcohol precipitation, the nucleic acids were divided into two equal portions. One half was used for a standard purification method employing CsCl centrifugation. The other was dissolved in FPLC buffer, treated with RNase A, and applied to a Superose 6 preparative grade column (HR 10/30). Plasmid eluted off the column within 20 min as a single, highly resolved peak. Plasmid isolated by FPLC had yields, purity, and transformation efficiencies similar to that isolated by CsCl centrifugation.     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Simultaneous purification of DNA topoisomerase I and II from eukaryotic cells

We have developed a procedure for the simultaneous purification of DNA topoisomerase I and II from calf thymus. Both enzymes were first extracted from isolated nucleoprotein complexes. After batchwise chromatography on hydroxylapatite the two enzyme activities were separated on a FPLC phenylsuperose column. The enzymes were further purified by a second chromatography on phenylsepharose (topo I) or FPLC Mono Q (topo II). The purification can be finished within three days, yielding 0.5-1.0 mg quantities of homogeneous, enzymatic active preparations of the two proteins from 200 g of starting material.     

Fractionation of charge-modified low density lipoproteins by fast protein liquid chromatography.

We describe a methodology developed to separate different forms of charge-modified low density lipoproteins (LDL) using the fast protein liquid chromatography (FPLC) system from Pharmacia. Lipoproteins were isolated by sequential ultracentrifugation and introduced onto an anion-exchange column (Mono Q HR 5/5). The multistep NaCl gradient elution was optimized and the analytical variables were determined on copper-oxidized LDL. After oxidation by copper for various times (up to 48 h), five forms were obtained (fractions A, B, C, D, and E). Within-run and day-to-day reproducibility were better than 8.6% and 10%, respectively. Protein and cholesterol recovery after the chromatographic separation was good (greater than 82%) and the detection limit was about 1 microgram. The more negative forms of collected LDL were mainly characterized by an increase in the lipid peroxidation product content, a depletion of vitamin E, an alteration of apoB and increased degradation by macrophages. The proposed methodology was applied to the study of LDL modifications generated by human umbilical endothelial cells and the protective effect of antioxidants (vitamin E and probucol).