Method For Large-scale Purification Research Articles

Purification and crystallization of benzoylformate decarboxylase.

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.

Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated with a novel large-scale purification method.

A novel, large-scale method for the purification of cytochrome-c oxidase from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The purified enzyme exhibited a heme a/protein ratio of 9.1 mmol/mg and revealed twelve protein bands after Tricine/SDS/PAGE. N-terminal sequencing showed that eleven of the corresponding proteins were identical to those recently described by Taanman and Capaldi [Taanman, J.-W. & Capaldi, R.A. (1992) J. Biol. Chem. 267, 22,481-22,485]. 15 of the N-terminal residues of the 12th band were identical to subunit VIII indicating that this band represents a dimer of subunit VIII (M(r) 5364). We conclude that subunit XII postulated by Taanman and Capaldi is the subunit VIII dimer and that cytochrome-c oxidase contains eleven rather than twelve subunits. We obtained the complete sequence of subunit VIa by Edman degradation. The protein contains more than 25% of charged amino acids and hydropathy analysis predicts one membrane-spanning helix. The purified enzyme had a turnover number of 1500 s-1 and the ionic-strength dependence of the Km value for cytochrome-c was similar to that described for other preparations of cytochrome-c oxidase. This was also true for the cyanide-binding characteristics of the preparation. When the enzyme was isolated in the presence of chloride, more than 90% of the preparation showed fast cyanide-binding kinetics and was resistant to formate incubation, indicating that chloride was bound to the binuclear center. When the enzyme was isolated in the absence of chloride, approximately 70% of the preparation was in the fast form. This high content of fast enzyme was also reflected in the characteristics of optical and EPR spectra for cytochrome-c oxidase purified with our method.

A highly purified antithrombin III concentrate prepared from human plasma fraction IV-1 by affinity chromatography.

We describe an improved method for large-scale purification of antithrombin III (AT-III) from human plasma involving heparin affinity chromatography of redissolved fraction IV-1 paste, viral inactivation by heating, followed by a second heparin affinity column. The characteristics of a new heparin affinity resin and the ability to extrapolate process behavior from small-scale (20 ml) to large-scale (40 liter) columns are described. This supports the use of the small-scale column for process optimization and validation studies in compliance with current regulatory requirements for biological products. The process has been characterized by analytical techniques including sodium dodecyl sulfate (SDS), reducing SDS, and nondenaturing polyacrylamide gel electrophoresis; laser desorption time-of-flight mass spectroscopy, and electrospray mass spectroscopy. These results demonstrate that greater than 95% of the protein in the final products is AT-III, which is greater than 95% active as defined by thrombin inhibition.

Dirofilaria immitis: A Large-Scale Purification Method and Partial Characteristics of a Superoxide Dismutase from Adult Worms

A superoxide dismutase (SOD) from adult worms of Dirofilaria immitis was purified using ethanol-chloroform and acetone treatment, DE 52 cellulose, and Sephadex G-75 gel chromatography to obtain a 677-fold purification and a specific activity of 5483 units/mg of protein. The purified SOD was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and composed of two identical 18.5 kDa subunits. The purified SOD was inactivated completely by 3 mM potassium cyanide, and reduced to about 18% of the initial activity by incubation at 100°C for 10 min and to 28% by treatment with 2% SDS. This means that D. immitis SOD differs markedly from the mammalian Cu/Zn SOD, although it is indeed a Cu/Zn SOD. Atomic absorption spectrometry revealed the presence of 0.80 mole of Cu and 0.78 mole of Zn per mole of subunit. The enzyme consisted of 22.6% acidic and 11.9% basic amino acids. No free amino acid was detected in the SOD by N-terminal amino acid sequencing.

5169772 Large scale method for purification of high purity heparinase from flavobacterium heparinum : Joseph J Zimmerman, Charles Cooney assigned to Massachusetts Institute of Technology

5169772 Large scale method for purification of high purity heparinase from flavobacterium heparinum : Joseph J Zimmerman, Charles Cooney assigned to Massachusetts Institute of Technology

Phosphorylation of tau protein by purified p34cdc28 and a related protein kinase from neurofilaments.

It has been suggested that hyperphosphorylation of the tau protein in neurofibrillary tangles may be relevant to the etiology of Alzheimer's disease and that at least one of the hyperphosphorylated sites lies within a consensus sequence for the p34cdc2/cdc28 family of kinases. We describe a new method for large-scale purification of p34cdc28 kinase from Saccharomyces cerevisiae and show that the purified enzyme can phosphorylate bovine and human tau. Phosphorylation was greatly enhanced by the addition of basic and acidic substrate modulators. The effect of the substrate modulators differed both with the structures of the substrates and the modulators. Similar results were obtained with a kinase that could be purified from neurofilaments by p13suc1 affinity chromatography, a hallmark of p34cdc2/cdc28-type kinases. These results are consistent with the hypothesis that a kinase of this type is involved in tau phosphorylation in vivo and open the possibility that hyperphosphorylation in Alzheimer's disease may be controlled by substrate modulators.

Large Scale Purification of Immunregulatory Peptide Nα1, N∊2-Diacetyl-Splenopentin by Chromatography on a Carboxylated Cation Exchanger

Abstract The immunregulatory pentapeptide ARG-LYS-GLU-VAL-TYR was synthesized by using the mixed anhydride coupling method. Acylation of Nα1- and N∊2-amino groups increases the enzymatic degradation stability of splenopentin. This paper describes a simple and effective large scale purification method of Nα1-, N∊2-diacetyl-splenopentin synthesis product by chromatography on the carboxylated cation exchanger Wofatit Y 79. Using ethanol/acidic water as eluent the isolation of purified peptide from the eluat was achieved directly by evaporation under vacuum and lyophilisation.

AMP Deaminase from Yeast: Role in amp degradation, large scale purification, and properties of the native and proteolyzed enzyme

Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools. This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4. Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734-8743). The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts. Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region. Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000. The unproteolyzed enzyme is highly unstable during purification. Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal. In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics. ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM. Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively. The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar. Thus, the N-terminal region is not required for catalysis or allosteric activation. AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP. The inhibition constants for these inhibitors decrease in the presence of ATP. ATP, therefore, tightens the binding of GTP, PO4, and AMP. The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism. Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators.

Improved method for large-scale purification of brain gangliosides by Q-sepharose column chromatography : Immunochemical detection of C-series polysialogangliosides in adult bovine brains

A new chromatographic method for separation of bovine brain gangliosides has been developed using Q-Sepharose. Gangliosides were separated based not only on their sialic acid numbers but also on the sialic acid molecular species and chain lengths of the skeletal oligosaccharide portions. The following results indicate that this column chromatography has practical advantages in separating mixtures of gangliosides, especially positional isomers and molecular species with N-acetyl- or N-glycolylneuraminic acid. (1) the loading capacity of Q-Sepharose for gangliosides was very high; (2) most major gangliosides such as GM1, GD1a, GD1b, GT1b and GQ1b were isolated in a single step; (3) these major gangliosides were clearly separated from gangliosides containing, N-glycolylneuraminic acid when examined using Hanganutziu-Deicher antibody; (4) polysialogangliosides that have four or more sialic acid residues were isolated efficiently. It was shown by the combination of Q-Sepharose column chromatography with thin-layer chromatography/enzyme immunostaining that adult bovine brains possess C-series polysialogangliosides as minor components which are known as embryonic molecules in avian and mammalian brains.

Properties of recombinant hepatitis B vaccine.

A large-scale purification method for hepatitis B surface antigen produced in a recombinant yeast (Saccharomyces cerevisiae) was established. The resulting HBsAg was greater than 99% pure and suitable for vaccine use. The yeast-derived HBsAg was structurally and biochemically similar to plasma-derived HBsAg. The anti-HBs antibody producing potency of the yeast-derived vaccine in mice was significantly higher than that of the plasma-derived vaccine. The yeast-derived vaccine induced protective antibody against hepatitis B virus of either adr or ayw subtype in a chimpanzee efficacy study. These observations demonstrate the usefulness of the yeast-derived vaccine as a second-generation hepatitis B vaccine.

Separation of the different microtubule-associated tau protein species from bovine brain and their mode II phosphorylation by Ca2+/phospholipid-dependent protein kinase C.

Bovine brain tau protein (tau) consists of four closely related phosphoproteins named tau 1, tau 2, tau 3 and tau 4, that range in size from 55 to 68 kDa (as determined by gel electrophoresis). Here we report an improved large-scale purification method for tau protein and the separation of the four individual tau protein species. The separation of the individual tau protein was accomplished by two chromatographic techniques: hydroxyapatite chromatography allowed the separation of two pairs of tau protein (tau 1 and tau 3) and (tau 2 and tau 4); fast protein liquid chromatography on a Mono Q column at basic pH achieved the resolution of the individual tau protein species in each pair derived from hydroxyapatite columns. Chromatography on the Mono Q column revealed that tau protein possesses previously unrecognized, highly reactive sulfhydryl groups that may oxidize to form intermolecular disulfide bridges. The isolation of individual species of tau in substantial quantities permitted an improved amino acid analysis that demonstrated the occurrence of cysteine and tryptophan in the protein. The availability of individual tau protein species greatly simplified the analysis for mode II phosphorylation of tau, which was found to be catalyzed by the calcium/phospholipid-dependent protein kinase C. The mode II phosphorylation of tau by protein kinase C was not associated with a mobility shift for tau protein in SDS-polyacrylamide gel electrophoresis, in contrast to mode I phosphorylation of tau by the Ca2+/calmodulin-dependent kinase, which produces a substantial shift in mobility.

Improved method for large scale purification of the phosphoenolpyruvate carboxylase of Escherichia coli K-12.

A rapid and simple method for large-scale preparation of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 was established. A strain carrying the ColE1-ppc+ (the gene of the enzyme) hybrid plasmid was used as an enzyme source. The cell-free extract was fractionated by ammonium sulfate, and the enzyme was purified to homogeneity by hydrophobic chromatography on hexyl-Sepharose using specific elution with L-aspartate, one of the allosteric effectors. The pure enzyme (153mg) was obtained from 137g wet cells in 4 days with 41% yield.

A rapid, large-scale method for purification of the metallo-carboxypeptidase inhibitor from potato tubers

A rapid, large-scale procedure has been developed for the preparation of metallo-carboxypeptidase inhibitor (CPI) from potatoes. The inhibitor is extracted with 80% ethanol from partially cooked potato tubers. After the ethanol is removed and the salts are dialyzed away, the inhibitor is isolated in pure form by chromatography on sulfopropyl Sephadex. Ten pounds of potatoes yields 250 mg pure inhibitor, representing over 40% recovery. Commercial mashed potato flakes contain about 0.02% carboxypeptidase inhibitor and can be utilized as a starting material in the procedure described to yield over 70 mg inhibitor from a pound of flakes.

The rapid purification of lactate dehydrogenase from alcaligenes eutrophus in a two-step procedure

A NAD-linked L(+)-lactate dehydrogenase (EC 1.1.1.27) was isolated from Alcaligenes eutrophus N9A. During purification advantage was taken of the high affinity of MatrexTM Gel Green A for this enzyme in crude extracts. One ml of this medium adsorbed 2660 U lactate dehydrogenase if 129 ml crude extract containing 2,480 mg protein were applied onto the column, as determined by frontal analysis. The enzyme was purified 275-fold by chromatography on this medium. Subsequent chromatography on Cibacron Blue F3G-A Sepharose 6B-CL resulted in a 3-fold purification and in a homogeneous preparation of lactate dehydrogenase. Starting with a crude extract containing 12 g total protein, the overall purification factor was 712.5, corresponding to a recovery of 36.1% activity and a specific activity of 776.6 U/mg protein. The affinity of MatrexTM Gel Green A medium to lactate dehydrogenase from other sources like A. hydrogenophilus, A. eutrophus A7, A. faecalis, Escherichia coli, Lactobacillus lechmannii, and rabbit muscle was investigated. Advantages of this method for large scale purification of lactate dehydrogenase from Alcaligenes eutrophus are discussed and compared to large scale purification methods applied for other enzymes.

Rapid and large-scale purification and characterization of renin from mouse submaxillary gland

Little is known about the structural basis for the highly restricted substrate specificity of renin, whose only function known to date is to hydrolyze the unique leucyl peptide bond in the prohormone angiotensinogen to form angiotensin I. Our lack of knowledge is due to our inability to purify this enzyme in a large quantity sufficient for structural studies. A two-step column chromatographic method for rapid and large-scale purification of renin from mouse submaxillary gland has been developed. It allows isolation of the enzyme in a hundreds-of-milligrams quantity at an overall yield of 60%. Single bands obtained by polyacrylamide gel electrophoresis, isoelectric focusing, and the result of ultracentrifugal studies indicated homogeneity of the product. The moelcular weight of renin was estimated to be 36,000 by sedimentation equilibrium studies in 6 m guanidine · HCl. Sedimentation velocity study gave a single sedimenting boundary with an s 20,w of 2.58 × 10 −13 s. A Stokes radius of 27 Å was obtained by gel filtration. The far ultraviolet-circular dichroism spectrum indicated a high content of β-structure (46%). In contrast to renal renin, submaxillary gland renin does not contain amino sugars or neutral sugars. No renin activity was retained by concanavalin Aagarose gels. Results of amino acid analysis, isoelectric focusing, and determination of amino-terminal residues by the dansylchloride reaction, together indicated that this protein is identical with renin A isolated previously in a much smaller quantity.

Purification and physicochemical characterization of human α 2hs-glycoprotein

A large-scale purification method for α 2HS-glycoprotein from normal human pooled serum is presented. 130 mg of α 2HS was obtained from 21 of normal human serum and the yield was 13.6%. Charge heterogeneity on isoelectrofocusing of this protein is mainly due to sialic acid. By the measurement of the circular dichroism spectrum, the α-helix content was calculated as 11% and the β-structure content was calculated as 21 to 33%. α 2HS consists of a single polypeptide chain (M r 49000) of which the N-terminal amino acid is alanine. The N-terminal sequence of 31 amino acids contains 19 hydrophobic residues.

Large-scale isolation of the two major polypeptides of the hepatitis B surface antigen (HBsAg) by gel filtration in 6 M guanidinium chloride

Purified hepatitis B surface antigen (HBsAg) of subtype ay was solubilized in guanidinium chloride and submitted to chormatography on Sepharose 4B in the presence of guanidinium chloride. The polypeptides P1 ( M r = 24,000) and P2 ( M r = 29,000) were eluted in the same fraction with a minor contaminant ( M r = 40,000). Large amounts of these two polypeptides were obtained in a single step. This technique which constitutes a method for large-scale purification of the P1 and P2 polypeptides should permit more complete characterization of the P1 and P2 polypeptides and of their antigenic determinants.

An improved method for large scale purification of human holo-transcobalamin II

25 mg of human holo-transcobalamin II with a specific cobalamin-binding capacity of 0.95 mol cobalamin/mol TC II was purified from 122 kg Cohn fraction III with a yield of 73% and a purification factor of 9.34 · 10 5. Consecutive purification steps comprised CM-Sephadex batchwise ion-exchange chromatography, affinity chromatography, using cyanocobalamin as a ligand, thermolabilly attached to 3.3′-diaminodipropylamine-substituted CH-Sepharose, and gel filtration. The high yield of the purification procedure was achieved by improving the stability of apo-transcobalamin II in the eluate of the CM-Sephadex, and by a few other modifications of a former procedure. In the latter, rapid denaturation of apo-transcobalamin II prohibited the use of long term affinity chromatography, which is obligatory for processing large amounts of Cohn fraction. In addition, subfractionation of transcobalamin II into smaller fragments which occurred in SDS-polyacrylamide gel electrophoresis in previous studies, was now reduced, indicating that proteolysis in the CM-Sephadex eluate had been prevented effectively.

Isolation of a soluble and template-dependent foot-and-mouth disease virus RNA polymerase

A large-scale purification method for the soluble RNA-dependent RNA polymerase from foot-and-mouth disease virus (FMDV)-infected BHK 21 cells is described. A cytoplasmic extract made 2.75 hr postinfection (fraction 1b) was chromatographed on DEAE-cellulose (fraction 2), poly(U)-Sepharose (fraction 3a, b) and sized on Ultrogel AcA 44 (fraction 4). Fraction 3a, b contained the viral protein P56 (the VIA antigen) plus small amounts of its precursor P72. The enzyme is capable of transcribing the synthetic template poly(A) primed with (Up) 5 U by a poly(U) polymerase activity. Furthermore, fraction 3a, b transcribed FMDV RNA with an RNA polymerase activity which did not require oligoribouridylate priming. These chromatographic procedures separated P72 from P56. P72 alone appeared to be transcriptionally inactive in vitro. Gel filtration of fraction 3a resulted in a high yield of pure P56. This fractionation step led to a considerable loss of transcribing activity.

A Rapid and Large-Scale Isolation of Renin from Mouse Submaxillary Gland by Pepstatin-Aminohexyl-Agarose Affinity Chromatography<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

A pressor enzyme, renin, was purified about 60-fold by chromatography on an affinity column including pepstatin-aminohexyl-agarose with a high yield of 83% from the homogenate of adult mouse submaxillary glands. The renin obtained by the one-step purification was electrophoretically homogenous on SDS-polyacrylamide gel and was as active as an absolutely pure renin. The renin purified by the affinity column could be separated into five active components by chromatography on CM-cellulose. Each of these renins gave a symmetrical elution profile on the CM-cellulose column and a discrete protein band on polyacrylamide gel electrophoresis at pH 8.6. Administration of nanogram quantities of each of the two major renin fractions to nephrectomized rats caused a sustained rise of blood pressure and decrease in sensitivity of the animal to angiotensin II. This rapid and large-scale purification method using pepstatin-aminohexyl-agarose eliminates all four fractionation steps reported previously for the isolation of mouse submaxillary gland renin.