A parallel affinity purification method for selective isolation of polyubiquitinated proteins

We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity-tagged ubiquitins by sequential use of affinity chromatography specific to each tag. In contrast with previous procedures using a single affinity-tagged ubiquitin, the PAP eliminates highly abundant ubiquitin monomers and monoubiquitinated proteins to selectively enrich proteins bearing both affinity-tags, or poly- and multiubiquitinated proteins. Accordingly, it would serve as a powerful method to facilitate mass-spectrometric identification of ubiquitinated proteins.     

A new method for high yield purification of type beta transforming growth factor from human platelets

A new method was developed for the purification of type beta transforming growth factor from human platelets. This method is a three-step procedure including gel filtration, weak cation exchange HPLC and reverse phase HPLC. All steps are carried out at low pH using exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by lyophilization facilitate the quantification of the growth factor in biological assays. Based on immunological characterization the purified acid-stable, highly basic transforming growth factor beta is the beta 1 form. Using the present method pure platelet TGF beta 1 is obtained in very high yield. 40 units of outdated human platelets yield 800 micrograms pure TGF beta 1, which is about a 10-20 fold higher yield than reported for other purification procedures.     

StreptoTag: a novel method for the isolation of RNA-binding proteins

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA-protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.     

A simplified method for purification of annexin V from human placenta

A simplified procedure for purification of annexin V from human placenta was developed. At first, the protein was separated from other proteins in membrane bound form in the presence of Ca2+, then was extracted with EDTA and purified by affinity chromatography on PAAG-immobilized phosphatidylserine. The purified protein gave a single band with a molecular weight of 35,000 in SDS-PAGE.     

Rapid purification of annexin V from human placenta by affinity chromatography

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.     

A new trophoblast-derived growth factor from human placenta: purification and receptor identification

This paper describes the identification and characterization of a new peptide growth factor. The peptide was isolated from trophoblastic brush border membranes of human placenta. The purified preparation was homogeneous and consisted of a single polypeptide of Mr 34 000 with a pI of about 6.0. This peptide stimulated DNA replication in cultured fibroblasts. The following association was seen between activity and protein: During DEAE-cellulose chromatography, both the 34-kilodalton (kDa) protein and the mitogenic activity displayed identical binding and salt dependence of elution. Nondenaturing electrophoresis at pH 8.3 revealed a comigration of the 34-kDa protein and the DNA replication stimulatory activity. Identical electrophoretic mobilities were displayed for both activity and protein at pH 7.0. These results demonstrate that the preparation is homogeneous and show that growth factor activity is intrinsic to the 34-kDa polypeptide. Binding of the 125I-labeled 34-kDa mitogen to target fibroblastic cells was specific; i.e., nanomolar concentrations of the unlabeled 34-kDa protein competed effectively with the labeled protein, whereas a variety of well-characterized growth factors and hormones were unable to compete even at micromolar levels. Thus the 34-kDa protein interacts with target cells through highly specific surface receptors. Chemical cross-linking techniques were used to investigate the identity of the receptor for the 34-kDa mitogen. Cross-linking of fibroblastic cells containing bound 125I-labeled 34-kDa protein generated a radiolabeled complex of 86 kDa in all four cell types examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oestrogen sulfotransferase: isolation of a high specific activity species from bovine placenta

During the course of a study of the control of expression of steroid-binding proteins in human mammary cancer oestrogen sulfotransferase was isolated from bovine placenta. By a combination of salt precipitation and ion-exchange and gel-permeation chromatography two forms of the enzyme were isolated. The forms, which apparently differ only in charge, have specific activities 100-300 times greater than has previously been reported for the enzyme. Partial peptide sequences of these enzymes are presented.     

An oligodeoxynucleotide affinity column for the isolation of sequence specific DNA binding proteins.

A nucleic acid affinity matrix containing a short oligodeoxynucleotide ligand has been prepared as an example of a material which can be used for the rapid and effective isolation of sequence specific DNA binding proteins. Two complementary oligodeoxynucleotides have been employed, one of which contains a small 5'-spacer arm with a terminal thiol group. Using this terminal thiol group, the ligand can be covalently coupled to Tresyl-activated Sepharose 4B or Epoxy-activated Sepharose 6B via a thioether linkage. This approach allows the specific attachment of the nucleic acid ligand via its 5'-terminus to the insoluble matrix. The double stranded affinity material was obtained by annealing of the complementary DNA fragment. As an example, we have used an eicosomer affinity column containing the sequence d(GAATTC) for the isolation of the Eco RI restriction endonuclease. Using a single column, the enzyme could be isolated by eluting the column with a single step or multistep gradient of increasing salt concentration. The enzyme was purified to 75%-85% homogeneity with yields of 0.1 mg to 0.2 mg from 0.5 g of cell paste.     

Tandem affinity purification of functional TAP-tagged proteins from human cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.     

A high yield method for the isolation of sheep's liver cathepsin L

A method, giving twice the yield of the previous method, for the isolation of sheep's liver cathepsin L is described. The method uses three phase partitioning (TPP) in t-butanol/water/ammonium sulphate mixtures, followed by two chromatographic steps, at different pH values, in a single column of S-Sepharose.     

A simple procedure for isolation of microsomes from human placenta

A method for rapid isolation of human placenta microsomes, which does not require facilities for ultracentrifugation was described. Such microsomes were compared with microsomes prepared by conventional ultracentrifugation technique. Both microsomal preparations were tested for protein, RNA and phospholipid content as well as for sulphohydrolase activities and glucose-6-phosphatase activity. The degree of contamination with other subcellular particles were tested as well. The data presented showed that microsomal proteins precipitated at pH 5.3 may be conveniently used for preparative separation of microsomal enzymes.     

Evaluation of a method for high yield purification of largely intact mitochondrial DNA from human placentae

We developed, and quantitatively and qualitatively evaluated an easily reproducible method for high yield purification of mitochondrial DNA (mtDNA) from human placentae by mechanical tissue disruption, differential centrifugation of mitochondria, enzymatic digestion, phenol extraction and ethanol precipitation. Average mtDNA yields were 2.5 microg/g tissue (without an RNAse treatment step) and 1.5 microg/g tissue (with an RNAse treatment step). This mtDNA migrated as a 16.5-kb isolated band in agarose gels; it yielded fragments of expected sizes after digestion with restriction enzymes; it successfully served as a template in long PCR for amplification of mtDNA sequences, and hybridized to an mtDNA probe in a predictable fashion. MtDNA yields of this method were 10-fold higher than those of previously reported ones for mtDNA purification from freshly obtained human cells and tissues, with the advantage that more placental tissue can be obtained for mtDNA purification than other types of tissue, at lower cost, and with minimal or no ethical issues.     

A rapid method for the isolation of coagulation factor XII from human plasma

Zinc chelate affinity chromatography was used to develop a rapid, three-step procedure to isolate coagulation factor XII from human plasma. The first step was ammonium sulphate fractionction which gave a 2-fold purification and 90% recovery in the 25–50% saturation fraction. The second step was zinc chelate affinity chromatography which gave a 240-fold purification and 67.5% recovery. The third step was zinc chelate affinity chromatography again, but with the application of a pH gradient. The overall recovery of zymogen factor XII was 21.7% and the total purification was 1992-fold. The purified factor XII had an apparent molecular weight of 77 600 as determined by SDS-polyacrylamide gel electrophoresis and a specific activity of 50 units/mg on a clotting assay.     

Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts

Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with [3H]propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. [3H]hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of [3H]hTNF-beta to the cells. We conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.