Staphylococcal acid phosphatase: extensive purification and characterization of the loosely bound enzyme

Acid phosphatase of Staphylococcus aureus PS55 was eluted from the surface of these cells with 1.0 m KCl at pH 8.5 by gentle agitation at 25 C and was purified 44-fold (51% recovery) by two cycles of dialysis and gel filtration. The eluted enzyme which had a 280/260 (nm) absorbancy ratio of 0.71 required at least 0.5 m salt solution for solubilization; however, most of the purified product which had a 280/260 (nm) absorbancy ratio of 1.72 was soluble in dilute buffer solution [0.01 m tris(hydroxymethyl)aminomethane chloride, pH 8.5]. Purified acid phosphatase appeared homogeneous according to the criteria of gel filtration, starch-block electrophoresis, and analytical ultracentrifugation. In a starch block, migration was toward the cathode at pH 8.0. Maximal activity occurred at pH 5.2 to 5.3 and salt concentration had little effect on phosphatase activity up to 1.0 m KCl or NaCl. Progressive loss of enzymatic acitivity occurred at higher salt concentrations. Molecular weight of purified acid phosphatase was estimated to be 58,000.     

Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp. SIB1 in cold-adaptation.

A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 degrees C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 degrees C compared to that at 20 degrees C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 degrees C with a k(cat)/K(m) value of 0.87 micro m(-1) x s(-1). When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T(1) refolding assay at 10 and 20 degrees C, the protein exhibited higher activity at 10 degrees C with a k(cat)/K(m) value of 0.50 micro m(-1) x s(-1). These k(cat)/K(m) values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria.     

An optimized system for expression and purification of secreted bacterial proteins

In this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-c-myc eptiope fusion proteins. Without regard to their initial solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, purified under denaturing conditions by immobilized metal affinity chromatography, and refolded by rapid dilution into a solution containing only Tris buffer and sodium chloride. Following concentration on the same resin under native conditions, each protein was eluted for further purification and/or characterization. Preliminary studies on a test set of 12 secreted proteins ranging in size from 13 to 130 kDa yielded between 10 and 50 mg of fusion protein per liter of induced culture at greater than 90% purity, as judged by Coomassie-stained SDS-PAGE. Of the nine proteins further purified, analytical gel filtration chromatography indicated that each was a monomer in solution and circular dichroism spectroscopy revealed that each had adopted a well-defined secondary structure. While there are many potential applications for this system, the results presented here suggest that it will be particularly useful for investigators employing structural approaches to understand protein function, as attested to by the crystal structures of three proteins purified using this methodology (B.V. Geisbrecht, B.Y. Hamaoka, B. Perman, A. Zemla, D.J. Leahy, J. Biol. Chem. 280 (2005) 17243-17250).     

Tetanus Toxin and Antigenic Derivatives I. Purification of the Biologically Active Monomer

A procedure for the isolation of pure tetanus toxin in a lethal monomeric form was developed based on the extraction of whole cells and chromatographic techniques. A crude extract of toxin was obtained by hypertonic extraction of cells from a 72-hr culture of Clostridium tetani Massachusetts strain. The extract was precipitated with ammonium sulfate and further purified by sequential use of ion-exchange chromatography and gel filtration. The degree of purification obtained by the fractionation procedures was monitored by polyacrylamide gel electrophoresis. The pure toxin has an average specific activity of 150 x 10(6) mouse MLD per mg of N and 3,000 Lf per mg of N. Immunological purity was demonstrated by a single line on both immunoelectrophoresis and agar double diffusion. One band was obtained on polyacrylamide electrophoresis, as was a single symmetrical peak in the ultracentrifuge and on Sephadex G-100 chromatography. The pure protein has an absorbancy ratio (280/260 mmu) of 2.1 in phosphate buffer (pH 7.5).     

Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography

In this study we describe a novel method for purification of Arabidopsis thaliana dehydrins overproduced in Escherichia coli. The cDNAs corresponding to the four dehydrin genes RAB18, LTI29, LTI30, and COR47 were inserted into a bacterial expression vector under an isopropyl beta-d-thiogalactopyranoside (IPTG) inducible bacterial promoter. After IPTG induction all four proteins accumulated in high amounts. The recombinant proteins were efficiently purified to over 95% purity with a three-step purification scheme: heat fractionation, immobilized metal ion affinity chromatography (IMAC), and ion exchange chromatography. In this study we introduce the novel use of IMAC as an efficient purification method for native dehydrins. Characterization of the purified proteins was done by Edman degradation, mass spectrometry, reverse-phase chromatography, and analytical gel filtration under native and denaturing conditions. Yields of purified proteins were between 2.8 and 12.5 mg per liter of bacterial culture, sufficient for further biochemical studies.     

huGM-CSF(9-127)-IL-6(29-184)融合蛋白的复性及纯化研究

利用Q Sepharose H.P.离子交换柱层析在8mol/L尿素变性条件下对huGM-CSF(9-127)-IL-6(29-184）融合蛋白进行初步纯化,然后再利用Sephacryl S-200分子筛柱层析复性及纯化后获得目的蛋白,其纯度达到95％以上。该纯化方案成功地解决了稀释复性或透析复性产物在进行Q Sepharose H.P.离子交换柱层析时目的蛋白不稳定而沉积于柱上的问题,获得了较好的复性效果,复性率达到80％以上。使用该纯化方案,1天内便可基本完成重组蛋白的复性及纯化过程,而且也便于扩大。     

Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets

S-145 (5Z-7-(3-endo-phenylsulfonylamino-(2.2.1.)-bicyclohept -2-exo-yl) heptenoic acid) is a potent and selective antagonist for thromboxane A2/prostaglandin H2 receptor. Using this compound as an immobilized ligand for affinity chromatography and [3H]S-145 as a radioligand, we have purified the thromboxane A2/prostaglandin H2 receptor from the membranes of human blood platelets. The purification procedures consisted of solubilization of the receptor with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), affinity chromatographies on columns of S-145 affinity gel, wheat germ agglutinin agarose and red agarose, and repeated gel filtration high performance liquid chromatography on a TSK gel G-3000SW column. On the second gel filtration high performance liquid chromatography, the [3H]S-145 binding activity was eluted as a symmetrical peak which overlapped exactly with a peak of ultraviolet absorption at 280 nm. By these procedures, the receptor was purified about 8700-fold from the solubilized extract with a recovery of 6%. The final preparation showed a broad protein band at Mr 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maximally bound 19.2 nmol of [3H]S-145/mg protein with a Kd of 29.8 nM. The [3H]S-145 binding to the purified receptor was specifically displaced by several thromboxane A2/prostaglandin H2 analogues.     

Purification of the Caenorhabditis elegans transposase Tc1A refolded during gel filtration chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12-16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

Novel type of murein transglycosylase in Escherichia coli.

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.     

Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase

Previously, the purification of DNA methyltransferase from murine P815 mastocytoma cells by immunoaffinity chromatography was described (Pfeifer, G.P., Grünwald, S., Palitti, F., Kaul, S., Boehm, T.L.J., Hirth, H.P. and Drahovsky, D. (1985) J. Biol. Chem. 260, 13787-13793). Proteins that stimulate the enzymatic activity of DNA methyltransferase have been purified from the same cells. These proteins, which partially coelute with DNA methyltransferase from DEAE-cellulose and heparin-agarose, are separated from the enzyme during the immunoaffinity purification step. A further purification of the stimulating proteins was achieved by butanol extraction, DEAE-cellulose chromatography and gel filtration on Superose 12. Two DNA methyltransferase-stimulating protein fractions were obtained. SDS-polyacrylamide gel electrophoresis of one fraction showed a single polypeptide with a molecular mass of 29 kDa. The second fraction consisted of 5 or 6 polypeptides with molecular masses 78-82 and 51-54 kDa. The proteins stimulate both de novo and maintenance activity of DNA methyltransferase about 3-fold. They enhance the methylation of any natural DNA and of poly[(dI-dC).(dI-dC)] but inhibit the methylation of poly[(dG-dC).(dG-dC)]. The purified proteins do not form a tight complex with DNA methyltransferase; however, they bind both to double-stranded and single-stranded DNA. The sequence specificity of DNA methyltransferase is obviously altered in presence of these proteins.     

The use of R-looping for structural gene identification and mRNA purification.

A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules. RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences. These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt. The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code. We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means.     

Cloning, expression, purification and activation by Na ion of halophilic alkaline phosphatase from moderate halophile Halomonas sp. 593

We have succeeded in the cloning of alkaline phosphatase gene, haalp, from moderate halophile Halomonas sp. 593. A deduced amino acid sequence showed a high ratio of acidic to basic amino acids, characteristic of halophilic proteins. The gene product was efficiently expressed in Escherichia coli BL21 Star (DE3) pLysS, but in an inactive form. The purified recombinant HaALP was separated into four fractions by gel filtration. When they were dialyzed against 50 mM Tris-HCl (pH 8.0)/2 mM MgCl? buffer containing 3 M NaCl, one of these four fractions was activated to almost full activity. This fraction contained a folding intermediate that was converted to the native structure by the salt. Among the additional salts tested, i.e., KCl, KBr, LiCl, MgCl?, (NH?)?SO?, and Na?SO?, only Na?SO? was effective, suggesting the importance of Na ion.     

An improved purification procedure for rat liver lysosomal phospholipase A1

A purification procedure is presented for the isolation of lysosomal acid phospholipase A1 (PLA1) from livers of non-pretreated rats, in a high yield and purity. The purification starts from a crude mitochondrial-lysosomal fraction. PLA1 is solubilised and subsequently purified by chromatography on concanavalin A-Sepharose, by chromatofocusing, and by gel filtration. After chromatofocusing, the enzyme is already purified 50200-fold with a yield of 50%, and after gel filtration 56600-fold with a yield of 7%. Purified PLA1 exhibits a specific activity of approx. 8.2 mumol phosphatidylethanolamine (preferred substrate) hydrolysed per min per mg protein, and upon chromatofocusing an apparent isoelectric point of 5.3 Gel filtration of purified PLA1 suggests a molecular mass of about 29 kDa, whereas in SDS-PAGE two proteins of 27 kDa and 55 kDa (mass ratio about 1/2) were visualised.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Effective expression of fragments of a botulinum neurotoxin type A gene, coding for the L-chain and H-chain in E. coli, with formation of products causing protective immunity to administration of the toxin

Native Clostridium botulinum gene coding for type A neurotoxin has been used to construct recombinant derivatives coding separately for L and H polypeptide chains of the toxin. The gene derivatives have been cloned into an expression vector pET28b in E. coli BL21 (DE3) cells. The recombinant L and H proteins seem to be the major individual proteins after IPTG induction of the recombinant cells. Each of the proteins has been accumulated only in inclusion bodies. The recombinant L chain (but not H chain) has been successfully resolubilized. Each of the proteins contains six His residues on the N terminus which allows purification on Ni-agarose columns with high yield. No toxic effect has been observed for both L and H chains after injection of 10 micrograms of recombinant preparations purified from inclusion bodies. Moreover, the injection resulted in an increase in the titer of specific antibodies which protected mice from 1 DLM of type A native botulinum neurotoxin. Hence, the recombinant neurotoxin protein derivatives which are present in E. coli inclusion bodies can be a source of material for producing diagnostic and therapeutic sera against type A botulinum neurotoxin.     

Purification and some properties of the glycolipid transfer protein from pig brain

A glycolipid-specific lipid transfer protein has been purified to apparent homogeneity from pig brain post-mitochondrial supernatant. The purified protein was obtained after about 6,000-fold purification at a yield of 19%. Evidence for the homogeneity of the purified protein includes the following: (i) a single band in acidic gel electrophoresis, in sodium dodecyl sulfate-gel electrophoresis, (ii) a single band in analytical gel isoelectric focusing, (iii) exact correspondence between the glycolipid transfer activity and stained protein absorbance in the acidic gel electrophoresis, and (iv) coincidence between the transfer activity and protein absorption at 280 nm in gel filtration through Ultrogel AcA 54. The protein has an isoelectric point of about 8.3 and a molecular weight of 22,000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecular weight of 15,000 was calculated from AcA 54 gel filtration. The amino acid composition has been determined. The protein binds [3H]galactosylceramide but not [3H]phosphatidylcholine. Under the conditions used, 1 mol of the transfer protein bound about 0.13 mol of [3H]galactosylceramide. The glycolipid transfer protein-[3H]galactosylceramide complex was isolated by a Sephadex G-75 chromatography. An incubation of the complex with liposomes resulted in the transfer of [3H]galactosylceramide from the complex to the acceptor liposomes. The result indicates that the complex functions as an intermediate in the glycolipid transfer reaction. The protein facilitates the transfer of [3H]galactosylceramide from donor liposomes to acceptor liposomes lacking in glycolipid as well as to acceptor liposomes containing galactosylceramide.     

Automated purification of recombinant proteins: combining high-throughput with high yield

Protein crystallography, mapping protein interactions, and other functional genomic approaches require purifying many different proteins, each of sufficient yield and homogeneity, for subsequent high-throughput applications. To fill this requirement efficiently, there is a need to develop robust, automated, high-throughput protein expression, and purification processes. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli (E. coli). The first is a filtration separation protocol in which proteins of interest are expressed in a large volume, 800 ml of E. coli cultures, then isolated by filtration purification using Ni-NTA-Agarose (Qiagen). The second is a smaller scale magnetic separation method in which proteins of interest are expressed in a small volume, 25 ml, of E. coli cultures then isolated using a 96-well purification system with MagneHis Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins, about 8 microg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 microg to 45 mg per purification run, as well as strategies for optimizing these protocols.     

Specific ribonucleases involved in processing of tRNA precursors of Escherichia coli. Partial purification and some properties.

Ribonucleases O and Q, the two putative nucleolytic activities which we detected previously in the crude extract from a thermosensitive ribonuclease P mutant (TS241) of Escherichia coli and which were shown to function in the processing of tRNA precursors in vitro, were partially purified from the 1000000 x g supernatant fraction of E. coli Q13. In the course of purification of these enzymes, the total RNAs synthesized in the thermosensitive mutant at the restrictive temperature were used as the substrates and the activities were identified from disappearance or alteration of specific tRNA precursor molecules in polyacrylamide gel electrophoresis. The purified ribonuclease O preparation cleaved specifically the multimeric tRNA precursors at the spacer regions. The purified ribonuclease Q preparation removed, in accordance with the definition of this enzyme, extra nucleotides from the 3'-terminal ends of monomeric tRNA precursors. Some properties of these two nucleases were investigated. In addition to these nucleases, another exonuclease (tentatively designated ribonuclease Y) and ribonuclease P, a well-characterized endonuclease, were also purified. The sequential mode of the processing of tRNA precursors, originally observed in the cleavage reactions with the crude extracts in vitro, was supported by studies with the purified enzyme preparations.