Cloning, Expression, and Purification of the Uropathogenic Escherichia coli Invasin DraD

In this study we presented a very efficient expression system, based on pET30LIC/Ek vector, for producing DraD invasin of the uropathogenic Escherichia coli and a one-step chromatography purification procedure for obtaining pure recombinant protein (DraD-C-His₆). This protein has a molecular weight of 14,818 and calculated pI of 6.6. It contains a polyhistidine tag at the C-terminus (13 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. Also, we obtained specific antibodies against DraD invasin to develop tools for characterizing the expression and biological function of this protein. The amount and quality of DraD-C-His₆ fusion protein purified from E. coli overexpression system seems to be fully appropriate for crystallographic studies (soluble form), and for establishing role of the protein in bacterium (cultured cell line interaction and in the internalization process) and for obtaining rabbit polyclonal antisera (insoluble form).     

Type II secretory pathway for surface secretion of DraD invasin from the uropathogenic Escherichia coli Dr+ strain

The virulence of the uropathogenic Escherichia coli Dr(+) IH11128 strain is associated with the presence of Dr fimbrial structures and a DraD invasin which can act as a fimbrial capping domain at the bacterial cell surface. However, a recent study suggests that the DraD protein is surface exposed in two forms: fimbria associated and fimbria nonassociated (prone to interaction with the N-terminal extension of the DraE protein located on the fimbrial tip). The actual mechanism of DraD surface secretion is presently unknown. We identified a previously unrecognized type II secretory pathway (secreton) in the uropathogenic E. coli Dr(+) strain which is well conserved among gram-negative bacteria and used mainly for secretion of virulence determinants. An active secreton is composed of 12 to 15 different proteins, among which GspD functions as an outer-membrane channel to permit extrusion of proteins in a folded state. Therefore, we inactivated the pathway by inserting the group II intron into a gspD gene of the type II secretion machinery by site-specific recombination. DraD secretion by the E. coli Dr(+) and gspD mutant strains was determined by immunofluorescence microscopy (with antibodies raised against DraD) and an assay of cell binding between bacteria and HeLa cells. The specificity of DraD-mediated bacterial binding for the integrin receptor was confirmed by examination of the adhesion of DraD-coated beads to HeLa cells in the presence and absence of alpha(5)beta(1) monoclonal antibodies. The investigations that we performed showed that type II secretion in E. coli Dr(+) strains leads to DraD translocation at the bacterial cell surfaces.     

Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

The protein invasin expressed on the cell surface of the pathogenic bacteria Yersinia pseudotuberculosis mediates the entry of this bacterium into cultured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protein constructs consist of part of the outer membrane domain of the invasin protein, six proline spacers, and a decamer of random peptides flanked by cysteine residues (CX(10)C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designated as ESPEL (E. coli Surface Peptide Expression Library). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bacterial clones were identified whose binding to human cells was mediated by peptides expressed on the bacterial cell surface. Flow cytometric analysis showed that both the identified bacterial clones and these corresponding chemically synthesized peptides bound to human cells specifically. The techniques described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors.     

Observation of the intracellular behavior of recombinant Yersinia pseudotuberculosis invasin protein

In this study, we observed the intracellular behavior of recombinant invasin, a 103-kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST-fused invasin C-terminal portion protein (GST-INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin-mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, (125) I-uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA-insoluble (polymerized-actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial-free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.     

Cloning, expression, and purification of the His(6)-tagged thermostable beta-galactosidase from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme

In the previous study we cloned Pyrococcus woesei gene coding thermostable beta-galactosidase into pET30-LIC expression plasmid. The nucleotide sequence revealed that beta-galactosidase of P. woesei consists of 510 amino acids and has a molecular weight of 59, 056 kDa (GenBank Accession No. AF043283). It shows 99.9% nucleotide identity to the nucleotide sequence of beta-galactosidase from Pyrococcus furiosus. We also demonstrated that thermostable beta-galactosidase can be produced with high yield by Escherichia coli strain and can be easy separated by thermal precipitation of other bacterial proteins at 85 degrees C (S. D $$;abrowski, J. Maciuńska, and J. Synowiecki, 1998, Mol. Biotechnol. 10, 217-222). In this study we presented a new expression system for producing P. woesei beta-galactosidase in Escherichia coli and one-step chromatography purification procedure for obtaining pure enzyme (His(6)-tagged beta-galactosidase). The recombinant beta-galactosidase contained a polyhistidine tag at the N-terminus (20 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. The enzyme was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni(2+)-TED-Sepharose columns. The enzyme was characterized and displayed high activity and thermostability. This bacterial expression system appears to be a good method for production of the thermostable beta-galactosidase.     

Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.     

Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells

Mammalian cell receptors that promote entry of intracellular bacteria into nonphagocytic cells have not been identified. We show here that multiple members of the integrin superfamily of cell adhesion receptors bind the Y. pseudotuberculosis invasin protein prior to bacterial penetration into mammalian cells. Affinity chromatography of crude detergent extracts demonstrated that integrins containing the subunit structures alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 bound to immobilized invasin. Furthermore, phospholipid vesicles containing isolated integrin proteins were able to attach to invasin. Specificity for invasin binding to the identified integrin receptors was also demonstrated, as immunoprobing and phospholipid reconstitution studies showed that the alpha 2 beta 1 integrin, beta 2 chain integrins, and vitronectin receptor (alpha v beta 3) were not involved in cellular attachment to invasin.     

Biochemical characteristic of biofilm of uropathogenic Escherichia coli Dr+ strains

Urinary tract infections caused by Escherichia coli are very common health problem in the developed countries. The virulence of the uropathogenic E. coli Dr⁺ IH11128 is determined by Dr fimbriae, which are homopolymeric structures composed of DraE subunits with the DraD protein capping the fiber. In this study, we have analyzed the structural and biochemical properties of biofilms developed by E. coli strains expressing Dr fimbriae with or without the DraD tip subunit and the surface-exposed DraD protein. We have also demonstrated that these E. coli strains form biofilms on an abiotic surface in a nutrient-dependent fashion. We present evidence that Dr fimbriae are necessary during the first stage of bacterial interaction with the abiotic surface. In addition, we reveal that the DraD alone is also sufficient for the initial surface attachment at an even higher level than Dr fimbriae and that chloramphenicol is able to reduce the normal attachment of the analyzed E. coli. The action of chloramphenicol also shows that protein synthesis is required for the early events of biofilm formation. Additionally, we have identified reduced exopolysaccharide coverage in E. coli that express only Dr fimbrial polyadhesins at the cell surface with or without the DraD capping subunit.     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

Cloning, sequencing, and overexpression of genes for ribosomal proteins from Bacillus stearothermophilus

Although a low resolution model for the arrangement of the proteins of the small and large ribosomal subunits is known, a detailed mechanistic understanding of the function of the ribosome awaits a high resolution structure of its components. While crystals have been obtained of several ribosomal proteins from Bacillus stearothermophilus, determination of atomic resolution structures of these proteins is impeded by the difficulty of obtaining large amounts of native proteins for crystallographic or NMR studies. We describe here the cloning and overexpression in Escherichia coli of the genes for ribosomal proteins S5, L6, L9, and L18 from B. stearothermophilus. S5 is extremely toxic to E. coli when overexpressed, and we have taken advantage of a new tightly regulated expression system to obtain high yields (more than 100 mg of pure protein/liter of culture) of this protein. The B. stearothermophilus S5 produced in E. coli crystallizes, and the crystals are identical to those obtained from the native protein. The crystals diffract to 2-A resolution.     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

促进原核表达蛋白可溶性的研究进展

以大肠杆菌为代表的原核表达蛋白系统具有操作简单、周期短、成本低、表达量高等优点而成为获得外源表达蛋白的首选方案.但外源蛋白在原核宿主中往往以无生物活性的包涵体形式存在,这限制了原核表达系统的广泛应用.随着对蛋白折叠动力学、参与蛋白折叠的酶和分子伴侣等认识的不断深入,科学家们通过诱导条件优化、宿主细胞改造以及使用融合标签...     

Sequence, localization and function of the invasin protein of Yersinia enterocoiitica

The inv locus of Yersinia enterocolitica is sufficient to convert a non-invasive Escherichia coli K12 strain into a microorganism that is able to penetrate cultured mammalian cells. The nucleotide sequence of inv reveals an open reading frame corresponding to an 835-amino-acid protein that is homologous to the invasin protein from Yersinia pseudotuberculosis. A polyclonal antiserum elicited by a synthetic peptide corresponding to the C-terminal 88 amino acids of this open reading frame detected a unique 100 kD protein in cell lysates of Y. enterocolitica strain 8081 c and in an E. coli strain harbouring the cloned inv gene. This protein localized to the outer membranes of both microorganisms and was cleaved by low concentrations of extracellular trypsin. HEp-2 cells were shown to attach to surfaces coated with bacterial outer membranes containing invasin and this attachment was destroyed by treatment of the membranes with trypsin. Thus it appears that the invasin protein from Y. enterocolitica is able to mediate both attachment to and entry of cultured epithelial cells.     

Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.     

蓝藻抗病毒蛋白-N基因的克隆、表达、纯化及活性鉴定

蓝藻抗病毒蛋白-N(Cyanovirin-N,CVN)具有强效抗HIV及其他包膜病毒活性,该蛋白序列特殊,难以重组制备,在大肠杆菌细胞质中形成包涵体。本研究根据大肠杆菌密码子偏好性对CVN原始核苷酸序列进行优化,通过多次PCR合成SUMO-CVN的全长DNA序列,构建pET3c-SUMO-CVN重组表达质粒,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。通过对诱导剂浓度和诱导时间的优化,发现以0.5mmol/LIPTG在20℃诱导24h可获得最高表达,SDS-PAGE结果显示,SUMO-CVN为可溶性表达,表达量占菌体总蛋白的28%;经特异性的SUMO蛋白酶对融合蛋白进行酶切及两步Ni-NTA凝胶亲和层析可以得到纯度较高的重组CVN蛋白。ELISA结果表明,重组蛋白CVN与gp120蛋白有较高的亲和力。体外抗病毒活性实验表明,重组蛋白CVN在纳摩尔浓度具有很好的抗HSV-1和HIV-1/ⅢB活性;这为开发基于CVN的新型、高效抗病毒药物打下了基础。     

Production of engineered human pancreatic ribonucleases, solving expression and purification problems, and enhancing thermostability.

Human pancreatic ribonuclease, the homolog of bovine pancreatic ribonuclease, has a significant therapeutic potential. Its study has been hindered by the difficulty of obtaining the enzyme in a pure and homogeneous form, either from human source or using heterologous expression. Engineering of different variants of human pancreatic ribonuclease has allowed us to study and overcome some problems encountered during its heterologous production in an Escherichia coli system and its purification from inclusion bodies. The 5'-end region of the mRNA that encodes the enzyme is critical for obtaining high expression levels. The results also suggest the importance of the proline 50 residue in the recovery yields of human pancreatic ribonuclease. All the variants produced are pure and homogeneous. Their homogeneity has been demonstrated by cation-exchange and reversed-phase chromatography and by mass spectrometry analysis. Moreover, enhancement of human pancreatic ribonuclease thermal stability is observed when residues R4, K6, Q9, D16, and S17 are changed to the corresponding residues of bovine seminal ribonuclease.     

Expression, purification, and characterization of recombinant human beta-amyloid42 peptide in Escherichia coli

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Evidence indicates that abnormal processing and extracellular deposition of the beta-amyloid42 peptide, the longer form of proteolytic derivative of the transmembrane glycoprotein-amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Since it is convenient and economical to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify beta-amyloid42 using glutathione-S-transferase (GST) fusion system. beta-Amyloid42 gene was inserted into a vector pGEX-4T-1 to construct a GST-fusion protein. The fusion protein GST-beta-amyloid42, expressed in BL21 (DE3) strain, was purified with GSH-affinity chromatography followed by thrombin cleavage. The digested product was further purified with an additional GSH-affinity and a Benzamidine chromatography step. After cleavage and purification, the beta-amyloid42 moiety showed the expected size of 4.5 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blot. Moreover, the fibrillar recombinant beta-amyloid42 exhibited great aggregation activity and showed neurotoxicity on neuron cells in vitro. These results suggest that our method will be useful in obtaining a large quantity of recombinant beta-amyloid42 peptide for further physiological and biochemical studies.     

The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin.

An essential virulence attribute for Yersinia enterocolitica and Yersinia pseudotuberculosis is the ability to invade the intestinal epithelium of mammals. The chromosomal invasin gene (inv) has been cloned from both of these Yersinia species, and the Y. pseudotuberculosis invasin has been well characterized (R. R. Isberg, D. L. Voorhis, and S. Falkow, Cell 50:769-778, 1987). Here we constructed TnphoA translational fusions to the Y. enterocolitica inv gene to identify, characterize, and localize the inv protein product in Escherichia coli. The cloned Y. enterocolitica inv locus encoded a unique protein of ca. 92 kilodaltons when expressed in minicells. A protein of comparable size was detected in immunoblots by using monoclonal antibodies directed against the Y. pseudotuberculosis invasin. This protein, which we also refer to as invasin, promoted both attachment to and invasion of cultured epithelial cells. These two functions were not genetically separable by insertional mutagenesis. We determined that the Y. enterocolitica invasin was localized on the outer membrane and that it was exposed on the bacterial cell surface, which may have implications for how invasin functions to mediate invasion.     

Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli

Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.     

Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70

Plasmodium falciparum heat shock protein (PfHsp70) has been proposed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. However, the biochemical and chaperone properties of PfHsp70 have not been elucidated. The heterologous overproduction of P. falciparum proteins in Escherichia coli is problematic because of its AT-rich genome and the usage of codons that are rarely used in E. coli. In this paper, we describe the successful overproduction of (His)(6)-PfHsp70 in E. coli using the pQE30 expression vector system. Initial experiments with E. coli [pQE30/PfHsp70] resulted in the overproduction of the full-length protein and truncated derivatives. The RIG plasmid, which encodes tRNAs for rare codons, was engineered into the E. coli [pQE30/PfHsp70] strain, resulting in significant reduction of the truncated (His)(6)-PfHsp70 derivatives and improved yields of the full-length protein. (His)(6)-PfHsp70 was successfully purified using nickel-chelating Sepharose affinity chromatography and its biochemical properties were determined. The V(max), K(m), and k(cat) for the basal ATPase activity of (His)(6)-PfHsp70 were found to be 14.6 nmol/min/mg, 616.5 microM, and 1.03 min(-1), respectively. Gel filtration studies indicated that (His)(6)-PfHsp70 existed largely as a monomer in solution. This is the first study to biochemically describe PfHsp70 and establishes a foundation for future studies on its chaperone properties.