Expression and purification of FtsW and RodA from Streptococcus pneumoniae,two membrane proteins involved in cell division and cell growth,respectively

FtsW and RodA are homologous integral membrane proteins involved in bacterial cell division and cell growth, respectively. Both proteins from Streptococcus pneumoniae were overexpressed in Escherichia coli as N-terminal His-tagged fusions. Their membrane addressing in E. coli was demonstrated by cell fractionation and was confirmed for FtsW by immunolocalization. Recombinant FtsW and RodA were solubilized from membranes using 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO). The detergent was exchanged to polyoxyethylene 8 lauryl ether (C12E8) during one-step purification procedure by Co(2+)-affinity chromatography. This procedure yielded 50-150 microg protein per litre of culture. Both proteins are likely to be folded as they are resistant to trypsin digestion and could be incorporated into reconstituted lipid vesicles.     

A heterogeneous tag-attachment to the homodimeric type 1 photosynthetic reaction center core protein in the green sulfur bacterium Chlorobaculum tepidum

The 6xHis-tag-pscA gene, which was genetically engineered to express N-terminally histidine (His)-tagged PscA, was inserted into a coding region of the recA gene in the green sulfur bacterium Chlorobaculum tepidum (C. tepidum). Although the inactivation of the recA gene strongly suppressed a homologous recombination in C. tepidum genomic DNA, the mutant grew well under normal photosynthetic conditions. The His-tagged reaction center (RC) complex could be obtained simply by Ni(2+)-affinity chromatography after detergent solubilization of chlorosome-containing membranes. The complex consisted of three subunits, PscA, PscB, and PscC, in addition to the Fenna-Matthews-Olson protein, but there was no PscD. Low-temperature EPR spectroscopic studies in combination with transient absorption measurements indicated that the complex contained all intrinsic electron transfer cofactors as detected in the wild-type strain. Furthermore, the LC/MS/MS analysis revealed that the core protein consisted of a mixture of a His-/His-tagged PscA homodimer and a non-/His-tagged PscA heterodimer. The development of the pscA gene duplication method presented here, thus, enables not only a quick and large-scale preparation of the RC complex from C. tepidum but also site-directed mutagenesis experiments on the artificially incorporated 6xHis-tag-pscA gene itself, since the expression of the authentic PscA/PscA homodimeric RC complex could complement any defect in mutated His-tagged PscA. This method would provide an invaluable tool for structural and functional analyses of the homodimeric type 1 RC complex.     

Rapid High-Yield Purification and Liposome Reconstitution of Polyhistidine-Tagged Sensory Rhodopsin I

We have used Ni²⁺-affinity chromatography as a rapid and efficient method to purify a sensory rhodopsin I (SR-I) derivative containing six consecutive histidine residues at its C-terminus (His-tagged SR-I). The protein was expressed inHalobacterium salinariumby integrating the corresponding gene at the chromosomal bacterioopsin locus under the control of the bacterioopsin promoter. His-tagged SR-I retains native SR-I photochemical reactions in purified membranes and phototaxis signaling functionin vivo.Immobilized Ni²⁺-affinity chromatography of membranes solubilized in 1% lauryl maltoside provides a single-step purification of the protein to electrophoretic homogeneity (≥90% pure). The procedure yields 1.7 mg pure photoactive protein/liter of culture (60% efficiency). This yield combined with engineered overproduction of the protein provides at least 120-fold greater amounts than that of a previously reported multistep purification procedure, permitting structural and biochemical analysis previously not feasible. The purified protein in lauryl maltoside at pH 5.3 exhibits a visible absorption maximum at 587 nm characteristic of SR-I. Spectrometric titration reveals an alkaline-induced species at 550 nm previously observed with transducer-free SR-I in native membranes. A previously unreported structured absorption band at 400 nm, consistent with a deprotonated Schiff base, forms with the same pK_aas the 550-nm species. His-tagged SR-I reconstituted into phosphatidylglycerol proteoliposomes retains properties of transducer-free SR-I in native membranes: its flash-induced absorption difference spectrum is identical, its photochemical reaction cycle kinetics show a similar pH dependence, and it forms a photoactive 550-nm species under alkaline conditions. These results indicate His-tagged SR-I reconstituted in proteoliposomes is suitable for analyzing SR-I interaction with its transducer proteinin vitro.     

Expression of caltrin in the baculovirus system and its purification in high yield and purity by cobalt (II) affinity chromatography

Direct protein extraction from animals is the only approach available to obtain caltrin, calcium transport inhibitor. Here we report the expression and purification of caltrin, previously shown to hinder the influx of calcium into epididymal spermatozoa. Cloning of the caltrin gene into the pCDNA3.1 V5/His-TOPO vector and the subsequent ligation of the caltrin-His sequence into the transfer vector pBacPAK9 allowed the expression of recombinant caltrin using the baculovirus expression vector system (BEVS). Recombinant His-tagged caltrin was purified utilising both nickel (II)-nitrilotriacetic acid (Ni(2+)-NTA) and cobalt (II)-carboxymethylaspartate (Co(2+)-CmAsp) immobilised metal affinity chromatography (IMAC). Using the BEVS, caltrin-His was identified in the supernatant and in the cell lysate, suggesting that caltrin is a secreted protein. Based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot results, purified recombinant caltrin-His was ascertained to be approximately 14.5kDa. Purification under the Co(2+) system yielded significantly purer protein samples when compared to the Ni(2+) system. Furthermore, Co(2+) was observed to bind the recombinant caltrin-His protein with higher efficiency and specificity and to yield a higher total protein concentration. Collectively, our results indicate that the Co(2+) system would be a better approach for purifying caltrin-His proteins than the Ni(2+).     

Localisation of the PsbH subunit in photosystem II: a new approach using labelling of His-tags with a Ni(2+)-NTA gold cluster and single particle analysis

Photosystem II core dimers were isolated from the green alga Chlamydomonas reinhardtii by Ni(2+)-affinity chromatography exploiting a 6 x His tag located at the N terminus of the PsbH protein. This protein is predicted to have a single transmembrane helix. In order to identify the location of PsbH within the photosystem II complex, the His-tagged core dimers were labelled using a Ni(2+)-NTA gold cluster and subjected to electron microscopy and image analysis. This new method enabled us to identify the location of the labelled His tag by statistical analysis of electron micrographs of the gold-labelled photosystem II complex. Comparison of these data with electron and X-ray crystallographic analysis of photosystem II indicates that the N terminus of PsbH is close to the two transmembrane helices of cytochrome b(559). Our analysis suggests that this approach is a powerful method to locate specific proteins within multisubunit complexes like photosystem II when crystallographic analysis is of insufficient resolution to directly identify amino acid side-chains. Moreover, it can be combined with cross-linking studies, and here we demonstrate that PsbH is a near neighbour of PsbX, which is consistent with the latter subunit being located close to the alpha and beta-subunits of cytochrome b(559). However, cross-linking between PsbH and PsbW was not detected despite the fact that the latter cross-linked with the alpha-subunit of cytochrome b(559).     

Oxidation of bis(terpyridine)cobalt(II) chloride by cytochrome c peroxidase compounds I and II

The bis(terpyridine)cobalt(II), Co(terpy)2(2+), reduction of cytochrome c peroxidase compound I, CcP-I, has been investigated using stopped-flow techniques as a function of ionic strength in pH 7.5 buffers at 25 degrees C. Co(terpy)2(2+) initially reduces the Trp191 radical site in CcP-I with an apparent second-order rate constant, k2, equal to 6.0+/-0.4x10(6) M(-1)s(-1) at 0.01 M ionic strength. A pseudo-first-order rate constant of 480 s(-1) was observed for the reduction of CcP-I by 79 microM Co(terpy)2(2+) at 0.01 M ionic strength. The one-electron reduction of CcP-I produces a second enzyme intermediate, CcP compound II (CcP-II), which contains an oxyferryl, Fe(IV), heme. Reduction of the Fe(IV) heme in CcP-II by Co(terpy)2(2+) shows saturation kinetics with a maximum observed rate constant, k3max, of 24+/-2 s(-1) at 0.01 M ionic strength. At low reductant concentrations, the apparent second-order rate constant for Co(terpy)2(2+) reduction of CcP-II, k3, is 1.2+/-0.5x10(6) M(-1) s-1. All three rate constants decrease with increasing ionic strength. At 0.10 M ionic strength, values of k2, k3, and k3max decrease to 6.0+/-0.8x10(5) M(-1) s(-1), 1.2+/-0.5x10(5) M(-1) s(-1), and 11+/-3 s(-1), respectively. Both the product, Co(terpy)2(3+), and ferricytochrome c inhibit the rate of Co(terpy)2(2+) reduction of CcP-I and CcP-II. Gel-filtration studies show that a minimum of two Co(terpy)2(3+) molecules bind to the native enzyme in low ionic strength buffers.     

H(+)/Ca(2+) exchange driven by the plasma membrane Ca(2+)-ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin-affinity purification

The plasma membrane Ca(2+)-ATPase was purified from Arabidopsis thaliana cultured cells by calmodulin (CaM)-affinity chromatography and reconstituted in proteoliposomes by the freeze-thaw sonication procedure. The reconstituted enzyme catalyzed CaM-stimulated 45Ca(2+) accumulation and H(+) ejection, monitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediately reversed by the protonophore FCCP, indicating that it is not electrically coupled to Ca(2+) uptake, but it is a primary event linked to Ca(2+) uptake in the form of countertransport.     

Polypeptide elongation factor Tu from Halobacterium marismortui

A GDP-binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high-salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF-Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF-Tus, and by immunological studies. Like some other EF-Tus the native protein can be cleaved with trypsin without concomitant loss of GDP-binding ability. The molecular mass of this hEF-Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein possesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS-PAGE, are described.     

Buffers and oscillations in intracellular Ca2+ dynamics

I model the behavior of intracellular Ca(2+) release with high buffer concentrations. The model uses a spatially discrete array of channel clusters. The channel subunit dynamics is a stochastic representation of the DeYoung-Keizer model. The calculations show that the concentration profile of fast buffer around an open channel is more localized than that of slow buffers. Slow buffers allow for release of larger amounts of Ca(2+) from the endoplasmic reticulum and hence bind more Ca(2+) than fast buffers with the same dissociation constant and concentration. I find oscillation-like behavior for high slow buffer concentration and low Ca(2+) content of the endoplasmic reticulum. High concentration of slow buffer leads to oscillation-like behavior by repetitive wave nucleation for high Ca(2+) content of the endoplasmic reticulum. Localization of Ca(2+) release by slow buffer, as used in experiments, can be reproduced by the modeling approach.     

Expression and activity of the nucleotide-binding domains of the human ABCA1 transporter

The aim of this study was the expression and production in Escherichia coli of the nucleotide-binding domains (NBDs) of the human ABCA1 transporter, in a soluble, non-denatured form. To increase the protein solubility, and avoid expression in E. coli inclusion bodies, we extended the length of the expressed NBD domains, to include proximal domains. The corresponding cDNA constructs were used to express the N-terminal His-tagged WT and mutant proteins, which were purified by Ni(2+)-affinity chromatography. Optimal expression of soluble proteins was obtained for constructs including the NBD, the downstream 80-residue domain, and about 20 upstream residues. The size homogeneity of WT and mutant NBDs was determined by Dynamic Light Scattering, and ATP-binding constants and ATPase activities were measured. The NBD1 and NBD2 domains bound ATP with comparable affinity. The ATPase activity of WT His-NBD1 was about three times higher than that of NBD2 and amounted to 5913 compared to 1979 nmol Pi/micromol NBD/min for WT His-NBD2. All engineered mutants had comparable ATPase activity to the corresponding WT protein. The optimisation of the length of the expressed proteins, based upon the boundary prediction of NBDs and neighbour domains, enables the expression and purification of soluble ABCA1 NBDs, with high ATPase activity. This approach should prove useful for the study of the structural and functional properties of the NBDs and other domains of the ABC transporters.     

The importance of calcium influx, calpain and calmodulin for the activation of CaCo-2 cell death pathways by Clostridium perfringens enterotoxin

CaCo-2 cells exhibit apoptosis when treated with low doses of Clostridium perfringens enterotoxin (CPE), but develop oncosis when treated with high CPE doses. This study reports that the presence of extracellular Ca(2+) in treatment buffers is important for normal activation of both those cell death pathways in CPE-treated CaCo-2 cells. Normal development of CPE-induced cell death pathway effects, such as morphologic damage, DNA fragmentation, caspase activation, mitochondrial membrane depolarization and cytochrome c release, was strongly inhibited when CaCo-2 cells were CPE-treated in Ca(2+)-free buffers. When treatment buffers contained Ca(2+), CPE caused a rapid increase in CaCo-2 cell Ca(2+) levels, apparently because of increased Ca(2+) influx through a CPE pore. High CPE doses caused massive changes in cellular Ca(2+) levels that appear responsible for activating oncosis, whereas low CPE doses caused less perturbations in cellular Ca(2+) levels that appear responsible for activating apoptosis. Both CPE-induced apoptosis and oncosis were found to be calmodulin- and calpain-dependent processes. As Ca(2+) levels present in the intestinal lumen resemble those of Ca(2+)-containing treatment buffers used in this study, perturbations in cellular Ca(2+) levels and calpain/calmodulin-dependent processes are also probably important for inducing enterocyte cell death during CPE-mediated gastrointestinal disease.     

Purification of human leukotriene C4 synthase from dimethylsulfoxide-differentiated U937 cells.

Human leukotriene C4 (LTC4) synthase was purified > 10000-fold from dimethylsulfoxide-differentiated U937 cells. Steps included: (a) solubilization of membrane-bound LTC4 synthase from microsomal membranes by the anionic detergent taurocholate; (b) successive anion-exchange chromatography steps in the presence of taurocholate plus Triton X-100 (primary anion exchange) then taurocholate plus n-octyl glucoside (secondary anion exchange); and (c) LTC2-affinity chromatography on a matrix that was constructed by first biotinylating synthetic LTC2 then immobilizing the biotinylated LTC2 on streptavidin agarose. The purification of human LTC4 synthase was enabled by the finding that LTC4 synthase activity in preparations enriched > 500-fold was absolutely dependent on the presence in LTC4 synthase incubation mixtures of divalent cations (specifically Mg2+) and phospholipids (specifically phosphatidylcholine), and that reduced glutathione, which was required at 2-4 mM for stabilization of LTC4 synthase, irreversibly inactivated the enzyme when present at > or = 5 mM during freeze/thaw cycles. The > 10000-fold purified LTC4 synthase preparation was comprised of three polypeptides having molecular masses of 37.1, 24.5 and 18.0 kDa. An 18-kDa polypeptide in both microsomal membranes and in the LTC2-affinity purified fraction was specifically labelled by a radioiodinated LTC4 photoaffinity probe (azido 125I-LTC4). The Km values in the LTC2-affinity purified preparation for reduced glutathione and LTA4 were 1.83 mM and 19.6 microM (respectively), closely resembling the Km values in isolated human blood monocytes. The Vmax of LTC2-affinity purified LTC4 synthase was 2-4 mumol LTC4 formed .min-1 x mg-1.     

Recombinant protein purification from pea

To assess the suitability of transgenic peas as a host for protein production from the perspective of ease of recovery, a strain containing recombinant beta-glucuronidase with poly(histidine) tail (GUSH6) was evaluated for solubility of the target protein in relation to native components (proteins, carbohydrates, and phenolics). Recovery of the recombinant GUSH6 from aqueous extracts by immobilized metal affinity chromatography with coupled Co(2+) yielded a nearly pure product with IDA (enrichment factor (EF) = 260) or NTA (EF = 200) resin. Single-step recoveries were also possible by isoelectric precipitation (EF = 4), polyelectrolyte precipitation (EF = 1.5), and anion-exchange chromatography (EF = 3.1), but enrichment factors were low.     

Cell chromatography: separation of different microbial cells using IMAC supermacroporous monolithic columns

Supermacroporous monolithic columns with Cu(2+)-IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross-linked with methylenebis(acrylamide) and has a network of large (10-100 microm) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild-type Escherichia coli (w.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. Wild-type E. coli and His-tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8-fold enriched with His-tagged E. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the Cu(2+)-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.     

Buffered non-fermenter system for lab-scale production of secreted recombinant His-tagged proteins in Saccharomyces cerevisiae

Expression of recombinant proteins using a secretion system can minimize co-purification of contaminating host proteins. Production of His-tagged recombinant proteins in the yeast alpha-factor secretion system has previously required a fermenter system to control the growth conditions such as pH of the yeast culture. We describe an inexpensive non-fermenter system for the production of secreted recombinant His-tagged proteins in Saccharomyces cerevisiae that uses a buffered low peptone YP glycerol medium, which does not interfere with immobilized metal affinity chromatography. Maspin, a tumor suppressor serpin, was expressed as a secreted N-terminal His/FLAG-tagged protein. Purification of the soluble active recombinant protein only requires centrifugation, concentration by ultrafiltration, and Ni2+ affinity chromatography. Purified protein yields of this system are 3-5 mg/L culture medium.     

Alternaric acid stimulates phosphorylation of His-tagged RiCDPK2, a calcium-dependent protein kinase in potato plants

Calcium-dependent protein kinases (CDPK) are an essential component of plant defense mechanisms against pathogens. We investigated the effect of alternaric acid, a host-specific toxin produced by the plant fungal pathogen Alternaria solani (Pleosporaceae), on a putative plasma membrane and cytosolic kinase RiCDPK2 of potato (Solanum tuberosum) and on hypersensitive cell death of host potato cells. Alternaric acid, in the presence of Ca(2+) and Mg(2+), stimulated in vitro phosphorylation of His-tagged RiCDPK2, a Ca(2+)-dependent protein kinase found in potato plants. We concluded that Ca(2+) and Mg(2+) play an important role in the interaction between alternaric acid and RiCDPK2. Based on our observations, alternaric acid regulates RiCDPK2 kinase during the infection process in an interaction between host and A. solani, leading to the inhibition of hypersensitive cell death in the host. We suggest that alternaric acid is a primary determinant by which A. solani stimulates CDPK activity in the host, suppressing hypersensitive cell death.     

The mechanism of uptake of cobalt ions by Neurospora crassa

Uptake of Co(2+) by 3-day-old mycelia of Neurospora crassa involves cell-surface binding as well as transport into the intracellular space. The surface binding is rapid and accounts for 30-40% of the total Co(2+) uptake. Transport of Co(2+) occurs at a rate of 40mug/h per 100mg dry wt. Surface binding and overall uptake show different temperature dependence. Metabolic inhibitors such as azide, dinitrophenol and fluoride depress transport of Co(2+). The overall uptake of Co(2+) exhibits a high K(m) value and hence the concentration mechanism is one of low ;affinity' for the metal. The uptake of Co(2+) varies linearly with pH in the range pH3 to pH6. Mg(2+) inhibits both surface binding and transport of Co(2+). It is suggested that the system that transports Mg(2+) is also involved in Co(2+) uptake by N. crassa.     

Phosphoglycerate mutase from Trypanosoma brucei is hyperactivated by cobalt in vitro, but not in vivo

Production of ATP by the glycolytic pathway in the mammalian pathogenic stage of protists from the genus Trypanosoma is required for the survival of the parasites. Cofactor-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shows no similarity to the corresponding enzyme in humans, and has also been genetically validated as a target by RNAi experiments. It has previously been shown that trypanosomatid iPGAMs require Co(2+) to reach maximal activity, but the biologically relevant metal has remained unclear. In this paper the metal content in the cytosol of procyclic and bloodstream-form T. brucei (analysed by inductively coupled plasma-optical emission spectroscopy) shows that Mg(2+), Zn(2+) and Fe(2+) were the most abundant, whereas Co(2+) was below the limit of detection (<0.035 μM). The low concentration indicates that Co(2+) is unlikely to be the biologically relevant metal, but that instead, Mg(2+) and/or Zn(2+) may assume this role. Results from metal analysis of purified Leishmania mexicana iPGAM by inductively coupled plasma-mass spectrometry also show high concentrations of Mg(2+) and Zn(2+), and are consistent with this proposal. Our data suggest that in vivo cellular conditions lacking Co(2+) are unable to support the maximal activity of iPGAM, but instead maintain its activity at a relatively low level by using Mg(2+) and/or Zn(2+). The physiological significance of these observations is being pursued by structural, biochemical and biophysical studies.