Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography

Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.     

Purification of antibodies to protein hormones by affinity chromatography on divinylsulfonyl sepharose

Antibodies to human growth hormone and ovine interstitial cell stimulating hormone have been purified from rabbit antisera by affinity chromatography using a newly developed divinylsulfonyl activated agarose. Elution of the antibodies was accomplished by neutral solutions containing chaotropic ions.     

Use of high salt sepharose 4B chromatography for the extraction of tRNA from plant tissues

tRNA has been isolated from Phaseolus seedlings, Parthenocissus cells grown in vitro and wheat germ by means of phenol extraction, ammonium sulfate precipitation of 25S and 18S ribosomal RNAs, retention on Sepharose CL4B in the presence of 1.85 M ammonium sulfate and stepwise purification on DEAE-cellulose. This method allows high recoveries of DNA-free tRNA with high accepting activity.     

Fractionation of Escherichia coli isoaccepting tRNA species by sepharose 4B column chromatography.

We have determined the elution profile on Sepharose 4B chromatographic column ofthe tRNA isoaccepting species of all 20 amino acids from Escherichia coli MRE 600. Further chromatography on a reversed phase column (RPC-5) is sufficient, in some cases, for a complete purification.     

Purification of microvillus membrane vesicles from pig small intestine by adsorption chromatography on sepharose

A simple, rapid method for the preparation of pure microvillus membrane vesicles from pig small intestine is described. The method is based on the ability of agarose beads to adsorb selectively the impurities, mainly basolateral membrane fragments, from a microvillus vesicle preparation isolated by hypotonic lysis, Mg²⁺ aggregation of contaminants and differential centrifugation.     

Purification of the sodium transport enzyme oxaloacetate decarboxylase by affinity chromatography on avidin sepharose

Aspartic acid can be covalently linked to yeast aspartyl-tRNA synthetase and to other proteins, in the absence of tRNA, under conditions where the synthetase activates the amino acid into aspartyl-adenylate, i.e., in the presence of ATP and MgCl₂. The linkage between aspartic acid and the protein is acid and alkali resistant; thus it is likely a peptide-like amide bond formed between the activated carboxylate group of aspartic acid and the primary amine function of the side chain of lysine residues.     

Purification of reticulocyte ribosomes using polyuridylic acid: sepharose chromatography.

Deoxycholate-KCl washed reticulocyte ribosomes are purified by affinity chromatography using Sepharose columns to which polyuridylic acid has been covalently bound. Upon passage through the column under nonenzymic conditions (high Mg²⁺:K⁺ ratio) approximately 10% of the ribosomes are retained. These ribosomes are then eluted with a buffer containing a high K⁺:Mg²⁺ ratio and are assayed for activity in various steps of the elongation process of protein synthesis. Approximately a 3- to 4-fold increase in the various activities compared with control ribosomes is obtained.     

Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength

A simple, rapid, and inexpensive method for the preparation and purification of chloroplast DNA (cpDNA) from pea has been developed. The crucial step is the isolation of chloroplasts in a medium of high ionic strength (I congruent equal to 1.40 M). CpDNA from pea prepared according to this method has successfully been used for restriction enzyme mapping, Southern transfers, and cloning.     

Purification of a special estrogen-binding protein from the liver of rats by affinity chromatography on estradiol sepharose

A procedure has been developed for purification of a special rat liver estrogen-binding protein. It includes protein precipitation by ammonium sulfate, gel filtration, ion exchange chromatography, and affinity chromatography on estradiol sepharose. The protein is purified 2260-fold with a 27% yield. Upon electrophoresis in 10% PAG in the presence of sodium dodecylsulfate the protein gives one polypeptide strip with a molecular weight of 31.200 +/- 400 dalton.     

Immobilization of polyribonucleotides in agarose gels at high ionic strength

Purine polyribonucleotides poly(A), poly(G), and poly(I) associate reversibly with agarose gels at high NaCl molarities over the pH range 6–10, at 20°?40°C. Pyrimidine polyribonucleotides poly (C) and poly(U) could not be immobilized in agarose gels under the above conditions. However, poly(C) could be immobilized in agarose without precipitation between pH 3.2 and 4.0. Association of poly(G) and poly(I) with agarose appears to decrease progressively with deprotonation of their purine residues, and both polymers interact with the gel very weakly above pH 10 regardless of NaCl concentration. The binding to agarose of these polymers at pH 7.5 is also strongly influenced by temperature in the range 20°?40°C. The association of single-stranded poly(A) is only shifted toward higher NaCl molarities by increased pH; its binding is also little affected by temperature in the above range. At NaCl molarities effecting the saturating retention in agarose and at neutral pH, the immobilization of several polynucleotides could be prevented by urea in a concentration-dependent manner. The corresponding profiles of urea molarity appear to disclose a number of hydrophobic interactions between polynucleotides and agarose, some of which could be relatively strong, especially in the case of poly(A).     

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.     

Archaea-based microbial fuel cell operating at high ionic strength conditions

In this work, two archaea microorganisms (Haloferax volcanii and Natrialba magadii) used as biocatalyst at a microbial fuel cell (MFC) anode were evaluated. Both archaea are able to grow at high salt concentrations. By increasing the media conductivity, the internal resistance was diminished, improving the MFC’s performance. Without any added redox mediator, maximum power (P _max) and current at P _max were 11.87/4.57/0.12 μW cm⁻² and 49.67/22.03/0.59 μA cm⁻² for H. volcanii, N. magadii and E. coli, respectively. When neutral red was used as the redox mediator, P _max was 50.98 and 5.39 μW cm⁻² for H. volcanii and N. magadii, respectively. In this paper, an archaea MFC is described and compared with other MFC systems; the high salt concentration assayed here, comparable with that used in Pt-catalyzed alkaline hydrogen fuel cells, will open new options when MFC scaling up is the objective necessary for practical applications.     

Affinity chromatography of galactose containing biopolymers using covalently coupled Ricinus communis lectin to sepharose 4B

A galactose-specific lectin isolated from Ricinus communis beans has been covalently coupled to Sepharose 4B activated with cyanogen bromide. The immonolized lectin retains its polysaccharide-binding property. The Sepharoselectin can be used for the purification of polysaccharides containing terminal nonreducing galactose.Only a small fraction of “native fetuin’ and ‘native ceruloplasmin’ are retarded on Sepharose-lectin. On analysis it was observed that hey had a lower content of sialic acid as compared to the native and unbound glycoproteins (sialated fractions). However, on desialation, fetuin and ceruloplasmin were completely adsorbed to Sepharose-lectin. The asialoglycoproteins interact strongly with Sepharose-lectin as compared to ‘partially sialated glycoproteins’. This has been attributed to the exposure of galactose residues of these glycoproteins on enzymatic desialation. These experiments demonstrated that Sepharose-lectin interacts with glycoproteins through their terminal, nonreducing galactose. On the basis of these experiments it is suggested that Sepharose-lectin can be used as an analytical tool for separation of ‘fully sialated glycoproteins’ from the ‘partially sialated glycoproteins’.