Purification and characterization of recombinant sTRAIL expressed in Escherichia coli

As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) has drawn considerable attention. This report presented the purification and characterization ofsoluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized andrefolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purifiedto electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purifiedsTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with EDs0 about 1.5mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had astructure similar to that of native protein with 13-sheet secondary structure. This efficient procedure ofsTRAIL renaturation may be useful for the mass production of this therapeutically important protein.     

Purification and characterization of recombinant spider silk expressed in Escherichia coli

A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk. Received: 7 April 1997 / Received revision: 24 July 1997 / Accepted: 25 August 1997     

Purification and characterization of recombinant human interleukin-29 expressed in Escherichia coli

Human interleukin (IL)-29 is the latest member of the class II cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-29, little is known of its functions in man. In the present study, an Escherichia coli expression system for the rapid expression of the human IL-29 gene was developed. It involved of cloning IL-29 gene into the pET-44 Ek/LIC vector, which allowed expression of IL-29 with a fusion tag consisting of the NusA protein, polyhistidine and S peptide (Nus-His-S-tag), and introducing a thrombin recognition site between the fusion tag and IL-29. The expressed fusion protein was purified by S-protein agarose affinity chromatography, and the fusion tag was removed from recombinant IL-29 by cleavage with thrombin. The purified IL-29 appeared a single band on SDS-PAGE, and the yield of IL-29 was 60 mg from 1 l of bacterial culture. N-terminal sequencing confirmed the identity of the purified protein. The recombinant IL-29 showed specific antiviral activity that was comparable to the commercially available IFN alfa-2b preparation.     

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.     

Overproduction, purification, and characterization of adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase, encoded by the purA gene of Escherichia coli, catalyzes the first committed step toward AMP in the de novo purine biosynthetic pathway and plays an important role in the interconversion of purines. A 3.2-kb DNA fragment, which carries the purA gene, was cloned into the temperature-inducible, high-copy-number plasmid vector, pMOB45. Upon temperature induction, cells containing this plasmid produce adenylosuccinate synthetase at approximately 40 times the wild-type level. A scheme is presented for the purification of the overproduced adenylosuccinate synthetase to homogeneity in amounts sufficient for studies of its structure and mechanism. The wild-type and the overproduced adenylosuccinate synthetase enzyme preparations were judged to be identical by the following criteria. The amino acid sequence at the N-terminus of the overproduced enzyme proved identical to the corresponding sequence of the wild-type enzyme. Michaelis constants for both the wild-type and overproduced enzyme preparations were the same. And (iii) both proteins shared similar chromatographic behavior and the same mobility during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results from size-exclusion chromatography and SDS-polyacrylamide gel electrophoresis suggest that adenylosuccinate synthetase exists as a dimer of identical, 48,000-Da, subunits.     

Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum

In the absence of the de novo purine nucleotide biosynthetic pathway in parasitic protozoa, purine salvage is of primary importance for parasite survival. Enzymes of the salvage pathway are, therefore, good targets for anti-parasitic drugs. Adenylosuccinate synthetase (AdSS), catalysing the first committed step in the synthesis of AMP from IMP, is a potential target for anti-protozoal chemotherapy. We report here the crystal structure of adenylosuccinate synthetase from the malaria parasite, Plasmodium falciparum, complexed to 6-phosphoryl IMP, GDP, Mg2+ and the aspartate analogue, hadacidin at 2 A resolution. The overall architecture of P. falciparum AdSS (PfAdSS) is similar to the known structures from Escherichia coli, mouse and plants. Differences in substrate interactions seen in this structure provide a plausible explanation for the kinetic differences between PfAdSS and the enzyme from other species. Additional hydrogen bonding interactions of the protein with GDP may account for the ordered binding of substrates to the enzyme. The dimer interface of PfAdSS is also different, with a pronounced excess of positively charged residues. Differences highlighted here provide a basis for the design of species-specific inhibitors of the enzyme.     

Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli

The high-level expression of human interleukin-1 beta in Escherichia coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion- and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N- and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1/LAF) assay. The specific activities determined with the IL-1/MCF and IL-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively.     

Purification and biochemical characterization of recombinant alpha 1-antitrypsin variants expressed in Escherichia coli

Site-directed variants of alpha 1-antitrypsin (alpha 1AT) expressed in a recombinant strain of Escherichia coli have been isolated with an overall process yield of 50% following tangential flow ultrafiltration, anion-exchange, immobilized metal affinity, and hydrophobic interaction chromatography. The primary structure of the purified variants including the integrity of the N- and C-termini has been verified by electrospray mass spectrometry of the intact molecules (44 kDa) for two of the variants (alpha 1AT Leu-358 and alpha 1AT Ala-357, Arg-358). Complementary classical peptide mapping and automated amino acid sequencing have verified 75% of the primary sequence of alpha 1AT Ala-357, Arg-358. Isoelectric focusing in an immobilized pH gradient revealed some microheterogeneity which proved to be reproducible from one purification batch to another. The isolated variants of alpha 1AT did not show any signs of proteolytic degradation during the purification process and proved to be fully active against their target proteases. The described process also allowed the complete removal of endotoxins from the preparations, opening the possibility to evaluate these novel protease inhibitors for their in vivo efficacy in different animal models of human disease.     

Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli

Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well with either E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.     

重组大肠杆菌右旋糖酐蔗糖酶的纯化及其性质

[目的]研究工程菌E.coli BL21(DE3)/pET28-dexYG产右旋糖酐蔗糖酶的纯化和酶学性质.[方法]工程菌经过IPTG诱导后生产含His-tag融合蛋白的右旋糖酐蔗糖酶,通过硫酸铵沉淀、Ni-NTA亲和层析纯化,得到纯度较高的酶蛋白,并对纯酶进行了酶学性质及动力学研究.[结果]经过SDS-PAGE测得该酶的分子量约为170 kDa,与理论推测值基本相同.以蔗糖为底物,酶促反应的最适温度为25～30℃,最适pH值为5.4,动力学常数Km值为10.43 mmol/L;酶活在pH 5.0～8.0较为稳定,在室温(25 ℃)保藏4天仍有59%的酶活力,4℃保存7周酶活力仅下降一半,但在35℃以上失活很快;Ca2 对催化作用有较大的促进,Mg2 有微弱的促进作用,K 对催化反应无影响,Cu2 的抑制作用最强.其他试剂对重组酶的活性有不同程度的影响,其中SDS抑制作用很强.[结论]研究为重组右旋糖酐蔗糖酶纯酶的获取、得到稳定性好、活性高的酶反应体系及利用该酶进行催化反应和工业化应用提供了重要参数.     

Purification of recombinant ribonuclease T1 expressed in Escherichia coli

A protocol for the rapid purification of ribonuclease T1 expressed from a chemically synthesized gene cloned into Escherichia coli is described. QAE ion-exchange and Sephadex G-50 chromatography are used to give over 300 mg (88% yield) of pure ribonuclease T1 from 61 of liquid culture in 3 days. We also report a new absorption coefficient for RNase T1: E1%278 nm = 15.4.     

Studies of ligand binding to Escherichia coli adenylosuccinate synthetase

Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined. These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme. Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding. The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands. TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site. Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate. These results suggest that the IMP and aspartate binding sites are spatially separated.     

Purification and characterization of a recombinant listeriolysin O expressed in Escherichia coli and possible diagnostic applications

The secreted pore-forming toxin listeriolysin O (LLO) is an essential virulence factor that allows the food-borne bacterial pathogen Listeria monocytogenes to escape from the phagocytic vacuole and reach the host cytosol. This protein belongs to the group of cholesterol-binding sulfhydryl-activated toxins, expressed by a large number of Gram-positive bacteria. A protocol for large-scale expression and purification of recombinant LLO was previously optimized. By a simple two-step purification method, we achieved a high-level LLO synthesis (4.5 mg l(-1) of cell culture) in a hemolytically active form (1.2 x 10(6) HU mg(-1) of protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures which is a difficult task, mainly owing to the low levels of toxin released in the culture media. Here we report the characterization of toxin properties and its preliminary application in an ELISA diagnostic test for listeriosis.     

Purification and characterization of aminoimidazole ribonucleotide synthetase from Escherichia coli

Aminoimidazole ribonucleotide (AIR) synthetase has been purified 15-fold to apparent homogeneity from Escherichia coli which contains a multicopy plasmid containing the purM, AIR synthetase, gene. The protein is a dimer composed of two identical subunits of Mr 38,500. The N-terminal sequence, amino acid composition, and steady-state kinetics of the protein have been determined. AIR synthetase has been shown to catalyze the transfer of the formyl oxygen of [18O]formylglycinamide ribonucleotide to Pi.     

Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli

Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro. The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared to CK2 from human. Kinetic measurements of the recombinant maize holoenzyme (rmCK2) revealed k(cat) values for ATP and GTP of 4 and 2s(-1), respectively; whereas the recombinant maize catalytic subunit showed almost equal values for ATP and GTP, i.e., ca. 0.8s(-1). A comparison of the k(cat)/K(m) ratio between the maize holoenzyme and the catalytic subunit from CK2 maize shows that the incorporation of the catalytic subunit into the holoenzyme leads to a 14-fold activation in the case of ATP and 8-fold activation in the case of GTP. The maize holoenzyme is about 10 times more sensitive towards CK2 inhibitor heparin, on the other hand, it is stimulated only 0% by polylysine as compared to the human counterpart. The maize holoenzyme activity is more sensitive towards NaCl concentrations higher than those of rhCK2 and treatment with urea showed that rmCK2 holoenzyme was denatured more readily than the human holoenzyme.     

Purification and biochemical characterization of a recombinant Anopheles gambiae tryptophan 2,3-dioxygenase expressed in Escherichia coli

In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway. TDO converts l-tryptophan to N-formylkynurenine by catalyzing the heme-dependent oxidative opening of the substrate indole ring. Despite the central role exerted by kynurenines in the physiology of living organisms, only a few insect TDOs have been subjected to biochemical characterization in vitro. We performed a RT-PCR-based analysis of the tissue distribution of TDO mRNA in A. gambiae that revealed a ubiquitous expression of the gene, thus further underlining the importance of the enzyme in the mosquito biology. We developed an expression/purification procedure yielding pure and active recombinant A. gambiae TDO. Spectral analyses showed that the enzyme was purified in its heme-ferric form that was subsequently used to determining the Michaelis-Menten constants of the TDO catalyzed reaction in the presence of reducing agents. The screening of a number of compounds as potential TDO modulators showed that several kynurenines and other Tryptophan-derived molecules interfere with the enzyme activity in vitro. Our study could contribute to understanding TDO regulation in vivo and to the identification of inhibitors to be used to alter Tryptophan homeostasis in the malaria vector.     

Purification and crystallization of Lactobacillus casei folylpolyglutamate synthetase expressed in Escherichia coli.

Folylpolyglutamate synthetase (FPGS) from Lactobacillus casei has been crystallized with polyethylene glycol and acetate buffer at pH 5.0. The enzyme was obtained from Escherichia coli strain SF4 harboring the L. casei FPGS chromosomal gene on a pEMBL vector (pGT3-8.1). Crystals of the enzyme were obtained which diffract to 2.6 A resolution. The crystals are monoclinic, space group P2(1), with unit cell dimensions of a = 54.07 A, b = 45.83 A, c = 84.37 A and beta = 107.92 degrees. A unit cell contains one molecule of the 43,000 Da enzyme per asymmetric unit. A complete X-ray data set on the native crystals has been collected.     

Preliminary X-ray crystallographic study of adenylosuccinate synthetase from Escherichia coli

Adenylosuccinate synthetase, a dimeric enzyme of 96,000 Mr, catalyzes the first committed step toward the de novo biosynthesis of AMP. Large, single crystals of adenylosuccinate synthetase from Escherichia coli grow from solutions of polyethylene glycol and ammonium sulfate. Crystals from ammonium sulfate belong to the orthorhombic space group P212121 with unit cell parameters a = 79.0 A, b = 70.2 A and c = 152.6 A. Crystals from polyethylene glycol belong to the space group P21, having unit cell parameters of a = 71.16 A, b = 71.99 A, c = 82.95 A, and beta = 71.52 degrees. The asymmetric units of both crystal forms probably contain the entire dimeric enzyme.     

Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19–405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni–NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 μg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 μg/ml.     

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for ¹⁵N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.