Preparative biochemistry最新文献

A simple and effective method has been developed to purify the recombinant protein tyrosine kinase pp60c-src from a baculovirus-insect cell expression system. The procedure includes affinity chromatography and HPLC. Milligram quantities of protein have been isolated with an activity of 3.9 mumol/min/mg protein using the substrate poly E4Y. This specific activity is many times higher than any published protocol. The enzyme is stable for months when stored in buffered 10% glycerol at -70 degrees C. This purification technique is compared to the immuno-affinity technique which is widely used for this enzyme. Enzyme kinetics were characterized with respect to substrate specificity, the effect of temperature, ionic strength, pH, and Mg+2 versus Mn+2 ions. Similar to the enzyme expressed in human cells, the recombinant enzyme demonstrated a higher Vmax and substrate specificity for poly E4Y over 5V-Agt-II. An activation energy of 14.2 kcal/mol was determined. Inhibition by increasing ionic strength is mostly due to an increase in Km for the poly E4Y substrate and hence was substrate dependent. The Km(ATP) was pH dependent while the Km(poly E4Y) was pH independent. For the phosphorylation of poly E4Y, free Mg+2 was stimulatory while Mn+2 was inhibitory. In contrast, Mn+2 stimulated the phosphorylation of 5V-Agt-II.

We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene (hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7-7, thereby creating a fusion gene construct (pT7-7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGP1-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of approximately 18% of total cell protein (approximately 50 mg/liter of bacterial culture). The fusion HisRS was purified to > 99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the functional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of M(r) = 53.7 kDa.

An immunosuppressive lectin was isolated from seed of Phaseolus vulgaris cv Cacahuate using physically entrapped stroma. The lectin was found to be a 94 kDa tetrameric protein. When 50 micrograms, of this lectin were administered intraperitoneally 2 days before the immunization with sheep red blood cells, humoral response against the immunogen was completely inhibited. Other properties of the protein are discussed.

Previously, a DNA-binding protein (DBPm) was identified in plant nuclei that may mediate the effects of DNA methylation on chromatin structure and transcription. In the present report, DBPm was partially purified from germinated pea (Pisum sativum) seed nuclear extracts by DEAE-cellulose, phenylsepharose, heparin-sepharose chromatography, and preparative mobility shift on polyacrylamide gels. The purified activity showed a band at approximately 50 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as by Sephadex G100 chromatography, suggesting that DBPm is present as a monomer.

Recent evidence suggests that polymorphonuclear leukocyte (PMN) elastase causes tissue injury in a variety of diseases. Current methods of purification of elastase involve several steps which result in a low yield. We report a simple purification method. PMN (10(9) in 4 ml of 0.05 M Tris, pH 7.8, containing 0.2% Triton X-100 were disrupted and homogenized by freezing and thawing followed by sonication. After centrifugation at 100,000 g for 20 min, enzyme was extracted from the pellet with 2.5 ml of 0.05 M Tris/1M NaCl (pH 7.8). The centrifugation-extraction cycle was repeated 3 times. Elastase from 10(8) PMN was then purified using a 1 ml Protease Inhibitor Affinity-Filter prepared by binding benzamidine to silica. Enzyme activity was determined by cleavage of the synthetic substrate N-Suc-(Ala)3-pNa. SDS-PAGE demonstrated 2 polypeptides, molecular masses of 29 and 27 kD with amino acid composition and partial N-terminal sequence (Ile-Val-Gly-Gly-Arg-Arg-Ala-Arg-Pro-His-Ala-Trp-Pro-) identical with those previously reported for elastase. We obtained 50 micrograms elastase (34-fold purification) with specific activity of 52 U/mg/min from 10(8) PMN. This represents a much greater recovery (23% yield) than is achieved by other methods. This method is simple, highly reproducible, and can be performed within a 2-day period.

Two kallikreins were identified in a homogenate of bovine pancreas in terms of their differential elution from an anion-exchange chromatography column. The kallikreins were quantified by their ability to release kinin from a partially purified preparation of bovine kininogen. The second kallikrein, designated kallikrein B, was purified by a three-step procedure following anion-exchange chromatography consisting of affinity chromatography on a benzamidine-agarose resin, gel filtration and hydrophobic interaction chromatography. An overall purification factor of 556-fold was achieved with a 58% recovery of enzymatic activity. The final material was shown to be homogeneous by a number of electrophoretic analyses. The relative molecular mass of pro-kallikrein B was found to be 26,700 by gel filtration and that of kallikrein B to be 26,000 by SDS gel electrophoresis. Gel isoelectric focusing revealed the presence of several isoenzymic forms ranging in isoelectric point from pH 4.05 to 4.35.

Eukaryotic initiation factors 2 and 2B (eIF-2; eIF-2B) are components of the rate-limiting step in the initiation of eukaryotic protein synthesis and are involved in the regulation of this process. When the alpha-subunit of eIF-2 is phosphorylated by an eIF-2 alpha kinase, the phosphorylated eIF-2 alpha (eIF-2 alpha(P)) binds tightly to eIF-2B and prevents the recycling of eIF-2.GDP to eIF-2.GTP which is required for sustained initiation of protein synthesis. The minute quantities of these proteins which are present in rat liver and muscle cytosol along with hundreds of other proteins has hindered purification efforts, as well as structure:function and regulatory studies. Therefore, procedures were developed for the simultaneous purification of eIF-2, eIF-2B and eIF-2 alpha kinase from kilogram quantities of fresh bovine liver. Briefly, the 0-45% ammonium sulfate precipitate of the 200,000 x g supernatant was solubilized and chromatographed on DEAE-cellulose, heparin-agarose, Mono Q, Mono S, and Superose columns. The availability of purified quantities of these factors will be useful for investigations of molecular mechanisms of action and antibody production.

Extract from the snake (Ptyas mucosa) pituitaries was capable of inhibiting the binding of 125I-labelled bovine growth hormone to female rat liver membranes. The growth hormone-like substance was not adsorbed on Concanavalin A-Sepharose nor DEAE-cellulose, but could be purified by gel filtration on Sephadex G-50. It possessed a molecular weight of about 19,000 as judged by SDS-polyacrylamide gel electrophoresis. The iodinated snake growth hormone-like substance bound to membranes prepared from female rat and pregnant rabbit livers. The binding could be inhibited by unlabelled snake growth hormone-like substance as well as by bovine growth hormone.

Because previous purification procedures for human kappa-casein may have caused the loss of some carbohydrate, relatively gentle methods were used. The protein was isolated by a four-step procedure which included isoelectric precipitation of whole casein, gel chromatography on Sephadex G-200 in the presence of SDS, removal of the SDS with Extracti-Gel D, and FPLC chromatography on Mono Q with buffers containing 6 M urea. The purified protein was nearly identical in amino acid composition to that found earlier by amino acid analysis and peptide sequencing and a molar extinction coefficient of 11.2 +/- 0.1 was determined on the basis of amino acid analysis with a norleucine internal standard. Hydrolysis, acylation, and methylsilylation of the carbohydrate, followed by gas chromatographic analysis on a fused silica column, yielded approximately 5% fucose, 17% galactose, 18% N-acetylglucosamine, 8% N-acetylgalactosamine and 7% sialic acid, totaling almost 55% by weight. The percentages from two different donors were almost the same. About 1 mole phosphorus per mole of kappa-casein was also detected. Using low-speed sedimentation equilibrium methods, a molecular weight of only 33,400 was obtained for human kappa-casein, suggesting carbohydrate lability. Human beta-casein with four phosphoryls was stabilized against precipitation by 10 mM Ca+2 ions at a level greater than 95% when the molar ratio of kappa/beta exceeded 0.15.

Affinity chromatography on immobilized Cibacron Blue (Matrex Gel Blue A) gel permeation chromatography on UltroPac TSK G 3000 SWG column and ion-exchange chromatography on "Mono Q" column were used to purify the malate dehydrogenase (MDH) from P. denitrificans to electrophoretic homogeneity. The last two purification steps were performed in FPLC system. The enzyme having a specific activity of about 2300 nkat/mg protein was obtained with an approximate 70% yield. MDH is a dimer with a molecular mass of 80,000 +/- 10,000 and an isoelectric point of 4.85 +/- 0.05. Absorption, fluorescence and CD-spectra were also measured and basic kinetic parameters were obtained for the homogeneous enzyme. The present paper also suggests the possibility of using the prepared enzyme for the determination of aspartate transferase (AST) in blood serum.