Purification of anthrax-toxin components by high-performance anion-exchange, gel-filtration and hydrophobic-interaction chromatography.

A procedure has been developed for purification of the tripartite anthrax-toxin components. This involves sequential high-performance anion-exchange, gel-filtration and hydrophobic-interaction chromatography. From an initial culture volume of 15 litres, typical yields of 8 mg of protective antigen, 13 mg of lethal factor and 7 mg of oedema factor are produced to higher degrees of purity than have previously been achieved by conventional chromatographic techniques.     

Purification and fractionation of lipopolysaccharide from gram-negative bacteria by hydrophobic interaction chromatography

By hydrophobic interaction chromatography on octyl-Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild-type smooth-form (S-form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S-form LPS according to the length of the O-polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O-polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re-mutant LPS contain four 3-hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S-form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re-mutant LPS a significant fraction of S-form LPS contains less than four 3-hydroxytetradecanoyl residues. Hydrophobic interaction chromatography also proved an effective one-step purification procedure of LPS as was shown with a crude preparation from S-form S. typhimurium.     

Adsorption chromatography of nucleic acids on silicone-coated porous glass

Bovine liver tRNA was adsorbed on silicone-coated porous glass in 5 M NaCl, 10 mM Tris-HCl (pH 7.6) and fractionated by elution with decreasing NaCl concentrations. tRNAPro, tRNAVal, tRNAIle, tRNAThr, tRNASer, and tRNAPhe were eluted in this order. tRNA which had been digested with ribonuclease A was not adsorbed. Q beta RNA (adsorbed onto the glass in 5 M NaCl) was eluted with 1.5 M NaCl. RNA species in a crude rRNA fraction from Escherichia coli were separated into tRNA, 5S rRNA, and high molecular weight rRNA on siliconized porous glass. A half of calf thymus DNA was adsorbed on the glass in 5 M NaCl and the residual part passed through the column. The CD spectra showed that DNA and tRNA took the C-form and the A-form in 5 M NaCl, respectively. Therefore, the discrepancies of behavior of the DNA and RNA on siliconized porous glass may be related to the occurrence of these forms. The recovery of these nucleic acids from the column was 83-100%. Adsorption chromatography on siliconized porous glass may be a useful method for the separation of tRNA, rRNA, and mRNA.     

Purification of a benzo[a]pyrene binding protein by affinity chromatography and photoaffinity labeling

Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29,000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30,000 was estimated by gel filtration chromatography of [3H]B[a]P- and [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 +/- 0.66 nM and a maximum number of binding sites of 2.07 +/- 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29,000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1-azidopyrene.     

High speed immuno-affinity chromatography on supports with gigapores and porous glass

Immuno-affinity chromatography exploiting the Ca²⁺ dependent interaction of the anti-Flag antibody and Flag-tagged proteins has been investigated. The antibody has been immobilized on porous glass beads (Prosep) containing gigapores and on a monolith, the polymethacrylate based Convective Interactive Media (CIM) column at a ligand density of 2 mg/g and 10 mg/ml respectively. The performance of the columns was assessed by applying clarified yeast culture supernatant containing overexpressed Flag-human serum albumin. Dynamic binding capacity and purity was checked at various flow rates ranging from 100 cm/h to 800 cm/h. 95% purity could be obtained. Anti Flag-CIM columns showed a higher unspecific adsorption, requiring a longer wash cycle to obtain the same purity compared to the Prosep column. Anti Flag-CIM columns showed a flow independent performance, which is explained by its monolithic structure. A decreasing dynamic binding capacity with flow was observed with anti-Flag-Prosep columns. Both columns are suited to purify milligrams of protein out of a yeast culture supernatant within a few minutes. We considered them as promising candidates for high throughput screening, where fast purification is a necessity.     

Purification and partial characterization of a novel binding protein for retinoic acid from neonatal rat

This report describes the purification and partial characterization of a novel retinoic acid-binding protein (CRABP-II) from neonatal rat pups. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-cellulose, and high performance liquid chromatography (HPLC) on a DEAE 5PW column. Two retinoic acid-binding peaks were resolved at the DEAE-cellulose step, with CRABP-I in the major peak and CRABP-II in the minor peak. Apparent homogeneity was achieved for both binding proteins after the HPLC step. CRABP-II consists of a single polypeptide, migrating with an apparent Mr of 15,000 in sodium dodecyl sulfate-polyacrylamide gel. It has an isoelectric point of 5.0. The dissociation constant for CRABP-II of retinoic acid was estimated to be 65 nM by fluorescence titration. Amino-terminal sequence analysis showed that CRABP-II has a distinct sequence, while the CRABP-I sequence is exactly identical to that of the rat testis CRABP. Despite the extensive sequence homology between CRABP-I and CRABP-II, antibodies directed against CRABP-I did not cross-react with CRABP-II.     

Characterization of the nontransformed and transformed androgen receptor and heat shock protein 90 with high-performance hydrophobic-interaction chromatography

The hydrophobicity of the nontransformed and transformed androgen receptor from rat submandibular gland and heat shock protein 90 (hsp90) from rat submandibular gland and liver was characterized by using high-performance hydrophobic-interaction chromatography on TSK gel Ether-5PW. In the absence of molybdate, cytosol [3H]R1881-androgen receptor complexes were mainly eluted in the 1.3 M region (Peak 1) with a small peak in the 0.8 M region (Peak 2) of a descending salt gradient (2 to 0 M) of ammonium sulfate. In the presence of molybdate, Peak 2 was predominant. When labeled-cytosol was applied after being heated at 25 degrees C for 30 min, a third peak (Peak 3) at around 0.64 M ammonium sulfate was newly observed. Peaks 2 and 3 were observed, while Peak 1 completely disappeared with the labeled-cytosol precipitated at 40% saturated ammonium sulfate. The Stokes radius of Peak 1 was 7 nm, and of Peak 2 was 8 nm. Both peaks were retained poorly by DNA-cellulose but bound rather well to DEAE-cellulose. These results suggest that these two peaks represent the nontransformed receptor, indicating that there are isoforms of the nontransformed androgen receptor which are distinguished by their hydrophobic properties and Stokes radii. Peak 3 had a Stokes radius of 5 nm and preferentially bound to DNA-cellulose, suggesting that this peak corresponds to the transformed receptor. These results indicated that the transformation of the androgen receptor accompanies the enrichment of the hydrophobicity of the receptor molecule. Hsp90 purified from rat livers and hsp90 in the cytosol both from livers and submandibular glands were eluted from Ether-5PW at 0.8 M ammonium sulfate, at almost the same position as Peak 2. This finding suggests that the enrichment of hydrophobicity on transformation is due to dissociation of hsp90 from the nontransformed androgen receptor.     

Amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) eggs

The complete amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) egg is presented. The 111-residue sequence was determined by the analysis of peptides generated by digestion of the S-carboxymethylated protein with Achromobacter protease I, chymotrypsin, or cyanogen bromide. The sequence is unique and not homologous to any known protein sequence. The protein may represent a new type of lectin.     

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography,partition and precipitation

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.     

Purification of chymotrypsin by subunit exchange chromatography on the denatured protein

The alpha-chymotrypsin subunits immobilized under denaturing conditions (6 M urea or 1% SDS) on CNBr-activated Sepharose 4B, were found to interact with soluble chymotrypsin subunits with the formation of oligomers higher than dimers. Subunits immobilized under nondenaturing conditions form only dimers. The effects of several parameters, such as organic solvents, cations, and anions of the lyotropic series, on the associating properties of the immobilized derivatives were examined. The interaction between immobilized and free enzyme was shown to be specific because extraneous proteins and compounds were not bound by the derivatives. Chymotrypsinogen, studied analogously, did not show appreciable self-associating capacity. Chymotrypsin subunits immobilized under denaturing conditions and packed in a column proved to be suitable for the purification of chymotrypsin from both bovine and porcine pancreatic extracts. The "subunit exchange" chromatography of such extracts, carried out between pH 2.5 and 4, gave an eightfold purification with a 93% recovery of chymotryptic activity. The specific activity was ca. 12,000 Schwert and Takenaka units/mg. Only 6% of the tryptic activity was bound by the column. The capacity of the matrix, 6 mg chymotrypsin/mL, dropped to about 70% of the original value after.     

Conformational changes of murine polyomavirus capsid proteins induced by sialic acid binding

Murine polyomavirus (Py) infection initiates by the recognition of cell membrane molecules containing terminal sialic acid (SA) residues through specific binding pockets formed at the major capsid protein VP1 surface. VP1 Pockets 1, 2, and 3 bind terminal SA, Gal, and second branched SA, respectively. The consequence of recognition on viral cell entry remains elusive. In this work, we show that preincubation of Py with soluble compounds within Pocket 1 (N-acetyl or N-glycolyl neuraminic acids) increases Py cell binding and infectivity in murine 3T6 fibroblasts. In contrast, Gal does not significantly alter Py binding nor infectivity, whereas sialyllactose, in Pockets 1 and 2, decreases cell binding and infectivity. Binding experiments with Py virus-like particles confirmed the direct involvement of VP1 in this effect. To determine whether such results could reflect VP1 conformational changes induced by SA binding, protease digestion assays were performed after pretreatment of Py or virus-like particles with soluble receptor fragments. Binding of SA with the VP1 Pocket 1, but not of compounds interacting with Pocket 2, was associated with a transition of this protein from a protease-sensitive to a protease-resistant state. This effect was transmitted to the minor capsid proteins VP2 and VP3 in virus particles. Attachment of Py to cell monolayers similarly led to a VP1 trypsin-resistant pattern. Taken together, these data present evidence that initial binding of Py to terminal SA induces conformational changes in the viral capsid, which may influence subsequent virus cell entry steps.     

A蛋白亲和色谱法纯化动物血清抗体的效果比较

比较Hitrap A蛋白琼脂糖凝胶亲和色谱系统,对不同动物血清或抗血清抗体的纯化效率,提供选择抗体纯化方法的依据。该方法简使快速,纯化的抗体纯度高,能较好地保持抗体免疫学活性。色谱柱反复使用40多次,纯化抗体效率不变。但A蛋白对不同动物血清的IgG吸附能力不同,对兔和豚鼠的血清抗体吸附能力强,而对小鼠、山羊和驴的血清抗体吸附能力较弱。说明虽然该方法纯化的抗体纯度高,但它对动物品种有选择。因此,应根据动物品种,选择适合的纯化方法。     

Influence of sialic acid on the binding activity of estrogen receptors

The influence of sialidase and sialyltransferase on the binding of 3H-estradiol to estrogen receptors in baboon uterus was investigated to ascertain if sialylation was involved. Specific binding capacity increased approximately 37% in the presence of sialidase, although Kd values essentially remained unchanged. 3H-Estradiol binding was correlated with free sialic acid in the presence of either sialidase or sialyltransferase. As sialidase concentrations were increased, 3H-estradiol binding and free sialic acid concentration increased linearly (r = 0.937, p less than 0.001). Incubation of 22 x 10(-5) U sialidase with its inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, decreased binding capacity and sialic acid concentration (r = 0.929, p less than 0.001). Although a decrease in binding capacity and free sialic acid concentration was observed in the presence of increasing amounts of sialyltransferase, a positive correlation was found between these two parameters (r = 0.839, p less than 0.035). A negative trend that was statistically insignificant was observed between binding capacity and sialic acid concentration when 2 x 10(-4) U sialyltransferase was incubated with the inhibitor, acetylsalicylic acid (r = -0.571, p = 0.195). The sialic acid concentration increased, while the 3H-estradiol binding capacity decreased. Collectively, these results show that both sialidase and sialyltransferase affect the binding of estradiol to its receptor in opposite directions. We suggest that biological activities of estrogen receptors in target cells may be regulated by the extent of sialylation of the receptor molecule itself. This posttranslational alteration may represent a new type of control mechanism for estrogen action.