Herpes simplex virus type 1 DNA polymerase. Mechanism-based affinity chromatography

The potent inhibition of herpes simplex type 1 (HSV-1) DNA polymerase by acyclovir triphosphate has previously been shown to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3'-end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism of inhibition of HSV-1 DNA polymerase has been used here to design an affinity column for the enzyme. A DNA hook template-primer containing an acyclovir monophosphate residue on the 3'-primer terminus has been synthesized and attached to a resin support. In the absence of added nucleotides, the column behaves as a simple DNA-agarose column, and HSV-1 DNA polymerase can be chromatographed using a salt gradient. The presence of the next required nucleotide encoded by the template (dGTP) increases the affinity of HSV-1 DNA polymerase for the acyclovir monophosphate terminal primer-template attached to the resin, and the enzyme is retained even in the presence of 1 M salt. The enzyme can be eluted from the column with a salt gradient after removal of the nucleotide from the buffer. Traditionally, the affinity purification of an enzyme relies on elution by a salt gradient, pH gradient, or more selectively by addition of a competing ligand (substrate/inhibitor) to the elution buffer. In the present example, elution of HSV-1 polymerase is facilitated by removal of the substrate from the buffer. This represents an example of mechanism-based affinity chromatography.     

Subunit protein-affinity isolation of Drosophila DNA polymerase catalytic subunit

gfLittle is known at present about the biochemical properties of very large-sized Drosophila DNA polymerases. In a previous study, we tried to purify Drosophila pol. catalytic subunit from embryos through seven column chromatographies and study its biochemical properties. However, we failed to characterize it precisely because an insufficient amount of the enzyme was generated. In this report, we describe direct purification from Drosophila embryos to near homogeneity using Drosophila DNA polymerase second subunit (Drosophila pol. 2) protein-conjugated affinity column chromatography and characterization of the enzyme in detail. To our knowledge this is the first demonstration of native DNA polymerase purification with activity using a subunit protein-affinity column. We observed new characteristics of Drosophila pol. catalytic subunit as follows: Drosophila pol. catalytic subunit synthesized DNA processively in the presence of both Mn(2+) and Mg(2+) ions, but Mn(2+) inhibited the 3'-5' proofreading activity, thereby decreasing the fidelity of DNA replication by 50%.     

Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase.

Herpes simplex virus-induced DNA polymerase purified by published methods was found to be contaminated with many others proteins, including virus structural proteins. Thus, DEAE-cellulose and phosphocellulose chromatography were used in combination with affinity chromatography to purify DNA polymerase from herpes simplex virus type 1- and type 2-infected cells. The purified enzyme retained unique features of the herpesvirus-induced DNA polymerase, including a requirement for high salt concentrations for maximal activity, a sensitivity to low phosphonoacetate concentrations, and the capacity to be neutralized by rabbit antiserum to herpesvirus-infected cells. By polyacrylamide gel electrophoresis, the purified DNA polymerase was associated with a virus-induced polypeptide of about 150,000 molecular weight.     

Transition state affinity jump chromatography. A double selection method for isolating catalytically active enzymes and other molecules

A double selection method for isolating active enzyme molecules, using substrate analog affinity chromatography and elution with transition state analogs, is described. To demonstrate the principle, a mixture containing native chymotrypsin and [3H]deoxychymotrypsin, in which the active site serine had been converted to [3H]alanine, was applied to a column containing immobilized D-tryptophan methyl ester. Both forms of chymotrypsin were retained. Catalytically active enzyme was selectively desorbed with the peptide aldehyde chymostatin, leaving catalytically inactive deoxychymotrypsin bound to the substrate analog affinity column. This affinity technique may afford a simple and general method for separating enzymes and other catalysts according to their molecular turnover numbers.     

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.     

Calf thymus DNA polymerase delta: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase alpha, a DNA dependent ATPase and proliferating cell nuclear antigen.

We have established a novel procedure to purify calf thymus DNA polymerase delta from cytoplasmic extracts. The enzyme has typical properties of DNA polymerase delta including a 3' - greater than 5' exonuclease activity and efficiently replicates natural occurring genomes such as primed single-stranded M13 DNA and single-stranded porcine circovirus DNA, this last one thanks to an associated or contaminating primase activity. A processivity of at least a thousand bases was evident and this in the apparent absence of proliferating cell nuclear antigen. The enzyme was purified through a procedure that allows the simultaneous isolation of DNA polymerase delta, DNA polymerase alpha-primase and a DNA dependent ATPase. All these enzymes coeluted from a phosphocellulose column. After chromatography on hydroxylapatite DNA polymerase delta separated from the coeluting DNA polymerase alpha and DNA dependent ATPase. Separation of the latter two was achieved on heparin-Sepharose. DNA polymerase delta was further purified by heparin-Sepharose and fast protein liquid chromatography. Purified DNA polymerase delta was resistant to the DNA polymerase alpha inhibitors BuPdGTP and BuAdATP and did not react with DNA polymerase alpha monoclonal and polyclonal antibodies. Based on this isolation protocol we can start to test biochemically the hypothesis whether DNA polymerase delta and DNA polymerase alpha might act coordinately at the replication fork as leading and lagging strand replicases, respectively.     

Method of purification of glucose oxidase by means of affinity chromatography on immunoabsorbent]

The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated.     

Purification of almond emulsin alpha-L-fucosidase I by affinity chromatography.

An affinity chromatographic method to purify α-l-fucosidase I from almond emulsin was developed. A derivative of lacto-N-fucopentaose II, ?-aminocaproyl-lacto-N-fucopentaosylamine, was coupled to sepharose 4B and packed in a column. By adopting this column for affinity chromatography, the enzyme was purified a hundredfold. The enzyme preparation was free from any other exoglycosidases which act on natural substrates.     

A rapid method for the purification of S-adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography

A simple method to purify S-adenosylmethionine: protein-carboxyl O-methyltransferase (protein methylase II, EC 2.1.1.24) from calf brain has been developed using affinity chromatography. The product of the reaction, S-adenosyl-l-homocysteine, which is a competitive inhibitor of the enzyme, was covalently linked to Sepharose beads. This gel proved to be an effective binder for protein methylase II at pH 6.2 and allowed for specific removal of the enzyme by the addition of the methyl donor substrate, S-adenosyl-l-methionine to the elution buffer. One step using this affinity chromatography column resulted in 377-fold purification of the enzyme and 71% recovery of the activity. Subsequent Sephadex G-100 chromatography enabled the enzyme to be purified 3000-fold from the calf brain whole homogenate. The purified enzyme showed a number of protein methylase II activity peaks following preparative gel electrophoresis with one major enzyme peak.     

Purification of acetate kinase by affinity chromatography.

A one-step procedure using affinity chromatography has been shown to purify to apparent homogeneity acetate kinase from a commercially available preparation and to partially purify the enzyme from a crude, cell-free extract. Since the gel's capacity for enzyme adsorption is controlled by the thermodynamics of ligand-enzyme interaction, maximization of the adsorption isotherm was attempted. Enzyme adsorption decreased logarithmically with increasing ionic strength but increased with increasing concentration of MgCl2. These competing effects caused the net adsorption of enzyme to increase to a maximum and then to decrease as the MgCl2 concentration was raised. The results allow a significant improvement in affinity column performance and have important implications for scale-up procedures.     

Spectroscopic properties of a novel neutral proteinase from Saccharomonospora canescens

Three distinct DNA polymerase fractions (A, B and C), were isolated from Trypanosoma cruzi epimastigote forms. Fraction A is a low molecular mass enzyme corresponding to beta-like DNA polymerase of T. cruzi. Fraction B co-purified along several purification steps with fraction A, but in the last step it was clearly separated by a phosphocellulose chromatography. Fraction C was separated from fractions A and B by binding to DEAE-cellulose column, since the other two fractions were eluted in the flowthrough. This enzyme has an apparent native molecular mass of 100 kDa and showed a high preference for poly(dC)-oligo(dG) among different template-primers tested as substrate. Western-blot and biochemical analysis strongly suggest that the three DNA polymerase fractions correspond to different molecular entities. These results are in agreement with the idea that fraction C is a new DNA polymerase of T. cruzi, not described before.     

用壳聚糖替代Sepharse 4B固定相关配体纯化酶的研究

采用壳聚糖固定酶作用的特定底物（菌体细胞）,并用戊二醛交联制备成酶的亲和吸附剂。采用该吸附剂纯化溶葡球菌的研究表明,经一步纯化可提高纯度4倍,酶活性回收大于70％。SDS－PAGE的电泳结果显示,产品基本上达到了标准酶的纯度。同时表明该吸附剂没有非特异性吸附。由载体壳聚糖替代Sepharose 4B制备的吸附剂具有简单、快速、较高收得率和操作安全等优点,适用于特定酶或基因工程产品的分离纯化。     

A rapid and efficient new method of purification of glutamate dehydrogenase by affinity chromatography on GTP-sepharose

The very high affinity for GTP of glutamate dehydrogenase was used to purify this enzyme by affinity chromatography. After periodic acid oxidation, GTP was covalently bound to an activated Sepharose. When crude mitochondrial extracts were applied on a column of this GTP-Sepharose, glutamate dehydrogenase was retained with very few other proteins. Glutamate dehydrogenase from rat liver was eluted with a KCl gradient with only one contaminating protein. From a pig heart mitochondrial extract the enzyme was purified 300-fold in one step. A chromatography on hydroxyapatite was sufficient to achieve the purification. This very simple technique avoids the long and troublesome crystallization steps generally involved in glutamate dehydrogenase purification.     

Interaction of Cibacron Blue F3GA with Escherichia coli DNA polymerase I and with T4 DNA polymerase.

Individual rapid procedures for the enrichment of Escherichia coli DNA polymerase I and of bacteriophage T4 DNA polymerase free of endonuclease activity are described using Blue dextran-Sepharose chromatography. The blue dye of Blue dextran-Sepharose selectively binds to the deoxynucleoside triphosphate substrate site of the E. coli but not the T4 enzyme indicating that the catalytic sites of these two enzymes which catalyze the same polymerization reaction in vitro are quite distinct.