Reassembly of a fimbrial hemagglutinin from Pseudomonas solanacearum after purification of the subunit by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Distilled water homogenates of Pseudomonas solanacearum B1, a highly fimbriated strain, strongly agglutinated human group A erythrocytes. The fimbriae and hemagglutinating activity were precipitated from the crude extract with 1% acetic acid, redissolved at pH 10, and precipitated again with 20 mM CaCl2 at pH 6.9. Ca2+, Mg2+, and Zn2+ had similar ability to precipitate the fimbrial hemagglutinin, but Na+ and K+ were much less effective. The fimbrial protein in the precipitate was purified to homogeneity by preparative gel electrophoresis in sodium dodecyl sulfate. The major protein band was eluted, and sodium dodecyl sulfate was removed by chromatography on ion retardation resin (AG 11A8) in 6 M urea. After dialysis against 10 mM sodium acetate (pH 4.5) to remove the urea, the protein reassembled to yield long fibers. These fibers were identical to fimbriae in the crude extract in diameter (6 nm) and in their ability to cause hemagglutination. The purified fimbriae contained no carbohydrates and wee similar to other bacterial fimbriae in amino acid composition, with hydrophobic amino acids comprising 41.8% of the total.     

A new method to precipitate myosin V from rat brain soluble fraction

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.     

Effect of zinc chloride and PEG concentrations on the critical flux during tangential flow microfiltration of BSA precipitates

There is renewed interest in the possibility of using precipitation for initial capture of high value therapeutic proteins as part of an integrated continuous downstream process. These precipitates can be continuously washed using tangential flow filtration, with long term operation achieved by operating the membrane modules below the critical filtrate flux for fouling. Our hypothesis was that the critical flux for the precipitated protein would be a function of the properties of the precipitate as determined by the precipitation conditions. We evaluated the critical flux using a flux‐stepping procedure for model protein precipitates (bovine serum albumin) generated using a combination of a crosslinking agent (zinc chloride) and an excluded volume precipitant (polyethylene glycol [PEG]). The critical flux varied with shear rate to approximately the 1/3 power, consistent with predictions of the classical polarization model. The critical flux increased significantly with increasing zinc chloride concentration, going from 60 L/m²/h for a 2 mM ZnCl₂ solution to 200 L/m²/h for an 8 mM ZnCl₂ solution. In contrast, the critical flux achieved a maximum value at an intermediate PEG concentration. Independent measurements of the effective size and viscosity of the protein precipitates were used to obtain additional understanding of the effects of ZnCl₂ and PEG on the precipitation and the critical flux. These results provide important insights into the development of effective tangential flow filtration systems for processing large quantities of precipitated protein as would be required for large scale continuous protein purification by precipitation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1561–1567, 2017     

Purification and structural characterization of recombinant rat gamma-interferon from Escherichia coli

An efficient method has been developed for the purification of recombinant rat gamma-interferon (rat rIFN-gamma). The procedure involves extraction of the Escherichia coli cell paste with 6 M guanidine-HCl (GuHCl), adsorption of the rat rIFN-gamma onto C8 alkyl-bonded silica, and elution with 50% propanol. The protein is essentially pure at this step, but is quantitatively precipitated by threefold dilution with aqueous buffer at pH 8.5. The precipitate is then dissolved with 6 M GuHCl in a buffer containing 0.05%. Tween-80 to about 0.3 mg/ml and dialyzed against the same buffer. The rat rIFN-gamma, which remains soluble on dialysis is again precipitated by dialysis against ammonium sulfate at 80% saturation. This final precipitate is readily soluble in 0.1 M ammonium acetate buffer, pH 8.5. The preparation is fully active and possesses a specific activity of 2-6 X 10(6) units/mg. The recoveries ranged from 50 to 85% in several experiments. The sequence of 20 amino acid residues from the NH2-terminus of the protein was determined using an automated sequencer and was found to agree with that deduced from the cDNA sequence.     

Precipitation of dilute chromatographic samples (ng/ml) containing interfering substances for SDS-PAGE

SDS-PAGE of chromatographic fractions requires prior removal of salts, detergents, denaturants, or organic solvents which may perturb the electrophoretic separation. Likewise, to successfully visualize minute amounts of protein present in chromatographic fractions, they must often be concentrated before analysis by SDS-PAGE. In this study, we used a dye precipitation procedure for simultaneous removal of interfering substances and concentration of dilute samples (ng/ml) before analysis by SDS-PAGE. Nanogram amounts of protein (143 ng) were effectively precipitated with a pyrogallol red-molybdate reagent from commonly used chromatographic buffers containing various interfering solutes or solvents. Proteins were successfully precipitated from solution in the presence of organic solvents (acetonitrile, methanol, 2-propanol), chaotropic agents (6 M urea, 6 M guanidine-HCl), a protein stabilizer (40% sucrose), metal chelators (30 mM EDTA and 30 mM EGTA), or high salt (1.0 M NaCl). Detergents, at concentrations up to twice their critical micelle concentrations, from the nonionic class (Triton X-100, Tween 20) or from the zwitterionic class (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) did not inhibit protein precipitation. Some interference was observed when proteins were precipitated in the presence of ammonium sulfate (0. 5-2.0 M). Proteins did not precipitate in the presence of ionic detergents (SDS and cetyltrimethylammonium bromide). The sensitivity of the combined pyrogallol red-molybdate precipitation/SDS-PAGE procedure is approximately 7 ng. Two other methods of precipitating proteins (trichloroacetic acid and phenol-ether) both exhibited varying degrees of effectiveness, ranging from 714 to 7 ng/ml, in the precipitation of individual proteins. In summary, the pyrogallol red-molybdate protein precipitation procedure facilitates the SDS-PAGE analysis of dilute protein samples (ng/ml) from chromatographic fractions of various compositions. The method is useful for rapid pilot-scale protein fractionation and facilitates the ongoing propensity of researchers to work with minuscule amounts of protein.     

The precipitation of mucin by aluminium

The interactions of Al with a mucin glycopeptide have been studied. A number of specific reactions were identified the nature of which were dependent upon the Al chemistry in the hydration environment. In particular, Al was observed to precipitate mucin and it is suggested that this proceeded via the intercalation of the hydroxide within the hydrated macroreticular network of the mucin biopolymer. This precipitation of mucin was visible by eye and abolished the viscosity of native mucin. Viscometry indicated that Al was bound by mucin at low pH. At pH > 3 Al formed a low molecular weight complex with mucin which was hydrolytically stable and was not precipitated at pH up to 8. In an additional and competitive reaction Al was bound by mucin and the resultant mucin–Al complex was suggested to be the precursor to self-assembled mucin–Al spheres identified in solution, by photon correlation spectroscopy, and in precipitate using selective histochemistry. The majority of these spherical structures were of sub-micron diameter and, through their interaction with each other, were probably responsible for the observed pH-dependent peaks of mucin solution viscosity. The larger spheres, between 20 and 80 μm in diameter, were only identified in isolated mucin/Al precipitates and, being comparatively rare, were unlikely to have influenced solution viscosities. These large spheres were observed to act as possible nucleation sites for the flocculation of mucin/Al precipitate. Al at concentrations as low as 0.015 mM induced changes in the rheological properties of mucin. Considering the ubiquitous nature of mucin and the degree to which it is conserved within biota the interactions of Al with mucin may have wide ranging implications for biological systems.     

The determination of the activity of immobilised amidases using an ammonia-sensitive glass electrode.

Ribosomal proteins, separated from rRNA by the LiCl-urea method, are then precipitated by adding one volume of 20% trichloroacetic acid. We show here that this precipitate can be dissolved directly into unbuffered Tris-OH for electrophoresis on acrylamide gels without further washing of the pellet or dialyzing of the solution.Ribosomal proteins can also be separated from rRNA by using 66% acetic acid in presence of 0.1 m Mg²⁺. We show here that the ribosomal proteins can be precipitated directly from the acetic acid by adding ten volumes of 10% trichloroacetic acid. This precipitate is then easily dissolved in unbuffered Tris-OH to prepare the proteins for acrylamide-gel electrophoresis without further treatment.     

Effect of egg yolk and other reagents upon the zinc of cold-shocked boar spermatozoa

A marked reduction (80.8%) in the zinc uptake by boar spermatozoa cooled to 4 degrees C occurs when the seminal plasma is pretreated with egg yolk-glucose at this temperature. Crude lecithin is less effective (59.8%). Similar pretreatment of the seminal plasma by the polycationic drug Antrypol, which totally removes the zinc-precipitable basic haemagglutinin, does not result in a significant reduction of the sperm zinc uptake at 4 degrees C.     

Characterization of a nutrient feed precipitate from an E. coli fermentation process

Metalloproteins require soluble metal ions such as zinc to properly fold into their native and active state to maintain stability and biological activity. When protein products are produced during microbial fermentations, metals are made available to the metalloproteins via nutrient supplements. During the production at the manufacturing-scale of a recombinant product that required zinc as a cofactor, an insoluble precipitate formed in the preparation tank after steam sterilization of the nutrient feed containing methionine, glycerophosphate, and zinc sulfate (MGZ). The precipitated nutrient feed was believed to be the cause for not enough zinc delivered to the production fermentor, leading to poor product assembly and stabilization. This article explores several analytical techniques such as capillary zone electrophoresis, inductively coupled plasma and phosphate molybdate assays to identify and quantify the composition of the precipitate. Our results show that the glycerophosphate component of the combined MGZ nutrient feed contains inorganic phosphate, which precipitates zinc from the feed media.     

Iron phosphate precipitation in Murashige and Skoog media

Murashige and Skoog revised medium, a standard plant tissue culture medium, precipitated on keeping because iron was ineffectively chelated. Most of the precipitate formed after two days and analysis indicated that it was predominantly iron phosphate. It was found that, after precipitation, the supernatant medium had lost c. 45% of its original iron, 20% zinc and 13% phosphate. The following modifications prevented precipitation: lowering the pH to below 3.2, increasing to 3 the molar ratio of EDTA:Fe or preparing the stock solution of FeEDTA with inadequate heating. Most of these modifications had a deleterious effect on the growth and greening of Ocimum basilicum suspension cultures. However, when an increase in the molar ratio of EDTA:Fe was achieved by reducing the iron concentration to one-third, there was no inhibitory effect on growth or greening of these suspension cultures.     

Stabilization of phosphofructokinase with sugars during freeze-drying: characterization of enhanced protection in the presence of divalent cations

Phosphofructokinase purified from rabbit skeletal muscle is fully inactivated after freeze-drying and dissolution. The addition of trehalose or maltose to the enzyme solution prior to freeze-drying results in a recovery of up to 80% of the original activity. Slightly less stabilization is imparted by sucrose, whereas glucose and galactose at concentrations up to 500 mM are relatively ineffective at protecting phosphofructokinase. Addition of ionic zinc to enzyme-sugar mixtures prior to freeze-drying greatly enhances the stabilization imparted by the above sugars. This effect is not simply due to the summation of the individual protective capacities of zinc and the sugar. Zinc alone affords no protection, but a high degree of stabilization is achieved when zinc is added to a sugar solution, even when the sugar is at a concentration at which, by itself, it is totally ineffective. In the presence of a constant sugar concentration (100 mM), freeze-dry stabilization of phosphofructokinase is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.9 mM) and the sugar concentration varied, the maximum stabilization is noted with less than 200 mM sugar. At higher solute concentrations the degree of enhancement decreases such that with 500 mM sugar the addition of zinc results in only a slight increase in protection. Several other organic solutes (proline, 4-hydroxyproline, glycine, trimethylamine N-oxide, glycerol and myo-inositol) that afford cryoprotection to phosphofructokinase, an effect enhanced by the addition of zinc, do not stabilize the enzyme during freeze-drying, even if zinc is present. The addition of ionic copper, cadmium, nickel, cobalt, calcium and manganese to trehalose-phosphofructokinase solutions prior to freeze-drying also increases the percentage of activity recovered after dissolution. Magnesium is ineffective in this respect.     

The relationship between reversibility of fibril formation and subunit composition of rat skin collagen

1. Salt-soluble rat skin collagen was precipitated from solution at neutral pH and 37 degrees . On cooling, a portion of the collagen returned into solution. The fractions were separated, the supernatant was concentrated and the precipitate was redissolved in dilute acetic acid. 2. Solutions of supernatant and precipitate were subjected to the same fractionation procedure, giving four fractions. 3. Each fraction was examined by starch-gel electrophoresis and a relationship between subunit composition and the fractionation procedure was noted. The collagen that redissolved on cooling contained less of the more highly cross-linked components than did either the fraction remaining in the precipitate or the starting material.     

Precipitation of copper using Desulfovibrio sp.

Sulphate-reducing bacteria isolated from submerged soil samples of paddy fields effectively precipitated copper from aqueous solution with maximum effect (75%) at 25 ppm Cu²⁺. As the copper concentration was increased to 100 ppm, precipitation efficiency decreased significantly. The use of bacteria to precipitate heavy metal ions from aqueous effluents is discussed.     

Sphingomyelin Favors. Precipitation of Negatively Charged Liposomes in 1 mM Ca2+

Abstract

Earlier studies showed that calcium and sphingomyelin increase in aortic intimal tissue with age. Liposomes having different aortic phospholipid concentrations were prepared to ascertain if the increase in calcium might have arisen from increased permeability to calcium. None of these liposomes were permeable to calcium, however, liposomes that contained sphingomyelin and carried an excess negative charge imparted by dicetylphosphate aggregatively precipitated out of suspension in 1 mM calcium chloride. The extent of precipitation was higher at higher sphingomyelin concentration but decreased as temperature increased. Cardiolipin with two negative charges per molecule supplied enough negative charge at 5 mol % to allow sphingomyelin containing liposomes to precipitate out of a 1 mM calcium chloride suspension. Precipitation also occurred when dipalmitoylphosphatidylcholine (with a transition temperature similar to sphingomyelin) replaced sphingomyelin in acidic liposomes.     

The precipitation of human IgG and its subunits with heparin

• 1.1. About 40% of IgG is precipitated when heparin is added to human plasma at pH 5.4, while only a little IgM and no IgA are precipitated.
• 2.2. The precipitation of purified IgG by heparin is pH-dependent and follows a sigmoid curve between pH 7.0 and 5.0.
• 3.3. The precipitate has a constant molar ratio heparin: IgG, independent of pH or the amount of heparin that is added.
• 4.4. The precipitate does not redissolve at high heparin concentrations.
• 5.5. The heavy chains of IgG precipitate also at pH 5.4, but this precipitate redissolves in excess heparin.
• 6.6. Light chains do not precipitate and the F_ab and F_c fragments are only partly precipitated.

     

Optimum conditions for the precipitation of chitosan oligomers with DP 5–7 in concentrated hydrochloric acid at low temperature

Chitosan was partially hydrolysed with 35% hydrochloric acid for 2 h at 80°C and the hydrolysate stored at −20°C after dilution with water to precipitate higher molecular weight (MW) chitosan oligomers. When the hydrolysate was not diluted with water, no precipitate was formed but 7.3% chitosan oligomers were precipitated at a dilution ratio of 1.0 (ml water/ml hydrolysate). The time for precipitation was not significantly changed after storing the hydrolysate at −20°C for 1 day. In addition, the precipitation yield was not significantly influenced by the concentration of HCl used for the hydrolysis except at less than 5.0 (ml HCl/g chitosan). However, the yield of precipitated oligomers changed with partial hydrolysis time. For 0.5 and 2 h hydrolysis, 10.1 and 7.3% of the oligomers were precipitated, respectively, but only 3.1% of the oligomers were obtained after a 4 h reaction. When methanol was added to the hydrolysate, the precipitation yield increased up to 70% but the amounts of lower MW chitosan oligomers in the precipitated oligomers also increased with the increase of higher MW. The precipitated oligomers were mainly composed of pentamers and hexamers.     

Escherichia coli primase zinc is sensitive to substrate and cofactor binding.

The ligation state of the single zinc site in primase from Escherichia coli changes when various substrates and cofactors are added alone or in combination as determined by X-ray absorption spectroscopy. X-ray absorption spectroscopy (XAS) provides information about the local structure (approximately 5 A) of atoms surrounding the metal and has been widely used to characterize metalloproteins. The zinc site in native primase and in primase bound to low (30 mM) magnesium acetate was found to be tetrahedrally ligated by three sulfurs at an average distance of 2.36 +/- 0.02 A and one histidine nitrogen located at a distance of 2.15 +/- 0.03 A. When ATP, ATP and (dT)17, or ATP, low magnesium acetate and (dT)17 was added to primase, one (or two) additional nitrogen/oxygen ligands were coordinated to the zinc together with the histidine nitrogen at an average distance of 2.15 +/- 0.03 A. These additional ligands are likely from adjacent phosphates from ATP. Another structure was observed for the primase-(dT)17 complex in which an additional nitrogen/oxygen ligand likely from the phosphate backbone together with the histidine nitrogen was located at a significantly shorter average distance of 2.05 +/- 0.03 A. High magnesium acetate (300 mM) completely inactivates primase in a reversible manner such that the region near the zinc ligands becomes accessible to proteolytic digestion [Urlacher, T. M., and Griep, M. A. (1995) Biochemistry 34, 16708-16714]. In this inactive complex, additional oxygen/nitrogen ligands from acetate as well as the histidine nitrogen are located at a distance of 2.20 +/- 0.03 A from the zinc site. To test whether the catalytic magnesium was binding within approximately 5 A of the zinc, we incubated primase with high (300 mM) manganese acetate. The functional properties of magnesium and manganese are similar, but the larger atomic number of manganese enhances the X-ray backscattering, making it possible to identify. Since no significant difference was observed from the manganese-incubated sample, the catalytic metal-binding site is likely located >5 A from the zinc. These studies clearly show that primase zinc ligation changes upon binding substrates.     

Characterization of Chinese hamster ovary cell culture feed media precipitate

Process intensification of monoclonal antibody production is leading to more concentrated feed media causing issues with precipitation of solids from the media solution. This results in processing problems since components in the precipitate are no longer in solution, changing the media composition and leading to variability in cell culture performance. The goal of this work is to characterize the feed media precipitate, and in particular to identify the precipitated components so that mitigation strategies can be developed. From the conducted analysis, the precipitate was predominately found to be organic and was analyzed with liquid chromatography-mass spectrometry and inductively coupled plasma-optical emission spectroscopy (ICP-OES) to identify the constituent components. Up to ten amino acids were identified with tyrosine (approximately 77 wt.%) and phenylalanine (approximately 4 wt.%) being the most prevalent amino acids. Elemental analysis with ICP-OES revealed that inorganic components were accounted for less than one weight percentage of the solid precipitate with metal sulfates being the predominant inorganic components.     

Ultrastructural and physiological studies on the longitudinal body wall muscle of Dolabella auricularia. II. Localization of intracellular calcium and its translocation during mechanical activity

The localization of Ca-accumulating structures in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia and their role in the contraction-relaxation cycle were studied by fixing the LBWM fibers at rest and during mechanical response to 400 mM K or to 10(-4)--10(-3) M acetylcholine in a 1% OsO4 solution containing 2% K pyroantimonate. In the resting fibers, electron-opaque pyroantimonate precipitate was mostly localized at the peripheral structures, i.e., along the inner surface of the plasma membrane, at the membrane of the surface tubules, and at the sarcoplasmic reticulum. In the fibers fixed during mechanical activity, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures. Electron-probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate may serve as a measure of Ca localization. These results are in accord with the view that, in the LBWM, the Ca stored in the peripheral structures is released into the myoplasm to activate the contractile mechanism.     

Expressing and purifying an anti-atherosclerosis polypeptide vaccine in Escherichia coli

A chimeric polypeptide of TTP-CETPC was successfully expressed as inclusion bodies in Escherichia coli by fusing it with the C-terminus of asparaginase and a basic amino acid-rich peptide (KR). After partially purified by washing with 0.5% (v/v) Triton X-100 in 10 mM PB, the pellet was solubilized in 8 M urea. The solution was precipitated with single volume and double volumes of cold ethanol for removing impurities. The fusion protein in solution was precipitated with triple volumes of ethanol to increase purity and then hydrolyzed with 50 mM hydrochloric acid at 55 degrees C for 72 h. The TTP-CETPC polypeptide was released after the unique acid-labile aspartylprolyl bond in the fusion protein was cleaved by acid. After impurities were removed by adjusting the hydrolysis solution pH to 9.45 and then to 8.37, the TTP-CETPC polypeptide was further purified by DEAE-cellulose column. The TTP-CETPC containing fractions were eluted at 60-80 mM NaCl. The purified TTP-CETPC cysteines were oxidized to form into intermolecular disulfide bonded dimers for immunizing mice. Specific anti-CETP antibodies in mice serum were assayed by ELISA and Western blot to verify that antibodies against CETP had been successfully induced and lasted for more than seventeen weeks in vivo.