Crystallization of a soluble,catalytically active form of Escherichia coli leader peptidase

Leader peptidase, a novel serine protease in Escherichia coli, catalyzes the cleavage of the amino-terminal leader sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Here, we report a procedure for the purification and the crystallization of a soluble non-membrane-bound form of leader peptidase (Δ2-75). Crystals were obtained by the sitting-drop vapor diffusion technique using ammonium dihydrogen phosphate as the precipitant. Interestingly, we have found that the presence of the detergent Triton X-100 is required to obtain crystals sufficiently large for X-ray analysis. The crystals belong to the tetragonal space group P4₂2₁2, with unit cell dimensions of a = b = 115 Å and c = 100 Å, and contain 2 molecules per asymmetric unit. This is the first report of the crystallization of a leader (or signal) peptidase. © 1995 Wiley-Liss, Inc.     

Abrin-a A chain expressed as soluble form in Escherichia coli from a PCR-synthesized gene is catalytically and functionally active

Abrin-a A chain (ABRaA) is a potent plant toxin, which possesses N-glycosylase activity toward eukaryotic 28S rRNA, and may have potential use in cancer therapy. To improve levels of expression in Escherichia coli, the gene encoding ABRaA was optimized by replacing rare codons with high-frequency ones, and synthesized using two-step PCR. The optimized ABRaA was cloned into the pET-His vector, and highly expressed in cytoplasm of E. coli. The yield of the purified recombinant (r) ABRaA proteins was up to 80 mg/l of induced culture. The rABRaA was one-step purified to homogeneity and its RNA-N-glycosylase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The MTT assay showed that it also had a killing effect on human hepatoma cell line SMMC-7721 and myeloma cell line Sp2/0. For the first time, ABRaA expressed as soluble form in E. coli from a PCR-synthesized gene is catalytically and functionally active.     

The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli

We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y. C., Wang, D. P., and Chiang, J. Y. L. (1990) J. Biol. Chem. 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver. By using the polymerase chain reaction, we have designed two P450c7 cDNAs. One has the second Met codon deleted and the third Thr codon replaced with an Ala. The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24). The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli. After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots. The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes. We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase. P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes. The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts. This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.     

Human rhinovirus 3C protease: cloning and expression of an active form in Escherichia coli

A cDNA encoding the viral protease from the 3C region of human rhinovirus type 14 was expressed in Escherichia coli through the use of a periplasmic secretion vector. The recombinant protease contained an eight amino acid N-terminal extension that enabled its detection by a specific antibody. It was expressed at a level of approximately 1 mg/L of E. coli culture. Biological activity of the protease was assessed in vitro by using a chemically synthesized peptide consisting of a consensus picornavirus protease cleavage site, Arg-Ala-Glu-Leu-Gln-Gly-Pro-Tyr-Asp-Glu. The peptide was cleaved by the recombinant protease at the Gln-Gly bond, generating the product peptides Arg-Ala-Glu-Leu-Gln and Gly-Pro-Tyr-Asp-Glu, which could be separated from the substrate peptide by reversed-phase HPLC. An in vitro assay for the rhinovirus 3C protease was developed by observing the rate of disappearance of the substrate peak from chromatograms of the supernatants of digestion mixtures.     

Purification and some properties of lysostaphin,a glycylglycine endopeptidase from the culture liquid of Staphylococcus simulans biovar staphylolyticus

This work presents a method for purification of lysostaphin, a glycylglycine endopeptidase, from the culture liquid of S. simulans biovar staphylolyticus to homogeneity in a few steps. The method includes ultrafiltration and ion-exchange and hydrophobic chromatographies. The enzyme was isolated in preparative amounts with the yield of 51%. Some physical and chemical properties of the enzyme are described.     

Expression in Escherichia coli of catalytically active phenylalanine ammonia-lyase from parsley

In parsley (Petroselinum crispum), phenylalanine ammonia-lyase (PAL) is encoded by 4 structurally similar genes. The nucleotide sequence of a near full-length cDNA and the deduced amino acid sequence of PAL-4 are presented and compared with the corresponding sequences of PAL-1, a previously described representative of the gene family. Transformation of Escherichia coli cells with PAL-1 or PAL-4 cDNA yielded catalytically active PAL, suggesting that the catalytic center of the enzyme is formed spontaneously rather than by a plant-specific mechanism.     

Overproduction of d-hydantoinase and carbamoylase in a soluble form in Escherichia coli

The production of d-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level of d-hydantoinase in an active form can be achieved at low temperature (25 °C) or by the coproduction of DnaJ/DnaK. Overall, with such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity. In addition, by fusion with thioredoxin, d-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK. These results indicate the success of our approaches to overproducing d-hydantoinase and carbamoylase in a soluble form in E. coli. Received: 26 November 1999 / Received revision: 28 February 2000 / Accepted: 10 March 2000     

Expression of rat heme oxygenase in Escherichia coli as a catalytically active, full-length form that binds to bacterial membranes

A plasmid, pKK-RHO, was constructed by incorporating the coding sequence of a cDNA for rat heme oxygenase into the expression vector pKK233-2. Escherichia coli strain XL1-blue transformed with pKK-RHO produced a catalytically active, full-length heme oxygenase. The 32-kDa native enzyme expressed, was localized in the bacterial membranes, possibly due to the spontaneous membrane-binding properties of a hydrophobic segment in its C-terminal region. During cultivation, a few degraded forms of heme oxygenase that had lost their membrane-associative properties appeared. Probably, some bacterial proteases cut the native heme oxygenase at sites near its C-terminus and so release hydrophilic peptides of heme oxygenase from the membranes. A 30-kDa polypeptide, one of the degraded forms of heme oxygenase, retained ability to accept electrons from NADPH--cytochrome P450 reductase and also activity for catalyzing breakdown of heme to biliverdin. The cultured cells were pale green. From them we extracted green pigment(s), of which the absorption spectrum closely resembled that of biliverdin, suggesting that a large amount of the endogenous heme of E. coli was actually degraded to biliverdin by the expressed heme oxygenase.     

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6 M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/μg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.     

Characterization of ribonuclease HII from Escherichia coli overproduced in a soluble form

Escherichia coli RNase HII is composed of 198 amino acid residues. The enzyme has been overproduced in an insoluble form, purified in a urea-denatured form, and refolded with poor yield [M. Itaya (1990) Proc. Natl. Acad. Sci. USA 87, 8587-8591]. To facilitate the preparation of the enzyme in an amount sufficient for physicochemical studies, we constructed an overproducing strain in which E. coli RNase HII is produced in a soluble form. The enzyme was purified from this strain and its biochemical and physicochemical properties were characterized. The good agreement in the molecular weights estimated from SDS-PAGE (23,000) and gel filtration (22,000) suggests that the enzyme acts as a monomer. From the far-UV circular dichroism spectrum, its helical content was calculated to be 23%. The enzyme showed Mn(2+)-dependent RNase H activity. Its specific activity determined using (3)H-labeled M13 RNA/DNA hybrid as a substrate was comparable to but slightly higher than that of the refolded enzyme, indicating that the enzyme overproduced and purified in a soluble form is more suitable for structural and functional analyses than the refolded enzyme.     

Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli

Hen egg white lysozyme was expressed as a protein fusion with the OmpA signal sequence and an octapeptide linker in Escherichia coli. The expression yielded soluble and enzymatically active lysozyme. Lysozyme activity was detected in the periplasmic space, in the cytosol and in the insoluble cytosolic fraction of E. coli. The results indicate that the environmental conditions in both the cytosol and the periplasmic space of E. coli were sufficient for correct protein folding and disulphide bond formation of eukaryotic recombinant lysozyme. However, the expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of the peptidoglucan. Correspondence to: B. Fischer     

Expression and Purification of Recombinant lysostaphin in Escherichia coli

Summary A 1.5 kb plasmid-encoded lysostaphin gene fragment of Staphylococcus staphylolyticus was amplified by polymerase chain reaction (PCR) and cloned in Escherichia coli by using plasmid pET29b(+) as an expression vector. By optimizing culture conditions, the activities of lysostaphin were expressed as 66 %, 30 %, and 4 % in extracellular, intracellular, and periplasmic fractions of recombinant E. coli, respectively. The enzyme was purified to homogeneity by using a simple one-step fractionation on bacterial cells of lysostaphin-resistant Staphylococcus aureus mutant. The recombinant enzyme had an Mr of approximate 27 kDa, and its bacteriolytic activity was indistinguishable to the authentic lysostaphin purified from Staphylococcus staphylolyticus.     

Expression of soluble and catalytically active plant (monocot) beta-glucosidases in E. coli

Complementary DNAs encoding mature beta-glucosidase proteins Glu1 and Glu2 of maize were amplified by the polymerase chain reaction (PCR) and cloned into the expression vector pET21a. Both Glu1 and Glu2 isozymes were expressed in high yield ( approximately 3.8% of the total soluble protein and 32% of the total expressed protein) in E. coli. Recombinant enzymes were active on a variety of artificial and natural substrates at levels similar to those of their native counterparts isolated from maize seedlings. Western blot analysis confirmed that both recombinant isozymes were immunoreactive with maize anti-beta-glucosidase sera and their molecular sizes were identical to those of the native maize Glu1 and Glu2 isozymes. Zymogram assays in native gels revealed that recombinant enzymes had the same electrophoretic mobility and substrate specificity as their native counterparts.     

Cytoplasmic and periplasmic expression of a synthetic gene for ferredoxin in Escherichia coli

A synthetic gene coding for a modified ferredoxin II of Desulfovibrio desulfuricans Norway strain was assembled from 10 oligonucleotides. This gene was cloned into various expression vectors allowing either cytoplasmic expression or export to the periplasmic space. In the latter case, two different constructs were made, each of which contained the OmpA signal peptide: one of these constructs contained 3 additional N-terminal amino acids as compared to the wild-type ferredoxin (56 amino acid residues). The expression of proteins encoded by the 3 constructs was assayed in E coli and the proteins were localized by cell fractionation and immunogold labelling. A low percentage of the periplasmic ferredoxin (approximately 5%) was secreted to the medium in the absence of cell lysis. The recombinant ferredoxin was purified and found to be correctly processed by the leader peptidase. However, due to the high cysteine content intramolecular and intermolecular disulfide bonds were formed and prevented binding of [4Fe-4S] clusters. Reconstitution experiments using these recombinant proteins are in progress.     

Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli

Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively high level in the cytoplasm (4 mg/L culture medium). Although the recombinant protein essentially accumulated as inclusion bodies, as much as 30% of the fusion protein was recovered in a soluble form at low growth temperature and could therefore be purified to homogeneity in a single-step procedure by metal-affinity chromatography. The recombinant precursor form of bovine carboxypeptidase A was recognized by a monoclonal antibody directed against purified bovine pancreatic carboxypeptidase A. Moreover, upon tryptic activation it gave rise to an enzyme, the N-terminal sequence, molecular size,and specific activity of which were comparable to those of the enzyme derived from the native precursor purified from bovine pancreas.     

Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate.     

High level expression of soluble angiogenin in Escherichia coli

Human angiogenin was genetically engineered and contained the E. coli Omp A signal sequence for secreting soluble angiogenin to the periplasm under tac promoter control. The angiogenin sequence was encoded in a single gene and expressed as a 14.4 kilodalton soluble protein in E. coli. It was purified by CM-Sepharose ion-exchange chromatography and by a heparin-Sepharose affinity chromatography procedure. The biological activity of angiogenin was established by its ability to inhibit mRNA-dependent rabbit reticulocyte cell-free translation.     

Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli

Lipase (EC 3.1.1.3) is a popular enzyme used as an ingredient in detergents and biocatalyst in many biochemical reactions. Lipase is usually expressed in Escherichia coli as an inactive inclusion body and at a low level. In this study, Candida antarctica lipase B (CalB) was fused with various polycationic amino acid tags and expressed in E. coli in order to increase a soluble expression level. By induction with 1.0 mM IPTG, the authentic and fused CalBs were expressed at 27-56% of total protein. The 10-arginine and 10-lysine tags fused at the C-terminal of CalB significantly increased the solubility of CalB by five- to ninefold, relative to the case of the authentic CalB expressed in a recombinant E. coli Origami 2? (DE3) strain. Among a series of the C-terminal poly-arginine tags, the recombinant CalB combined with the 10-arginine tag (CalB-R10) possessed the highest lipase specific activity of 9.5 ± 0.03 U/mg protein, corresponding to a fourfold enhancement compared with the authentic CalB.     

Direct expression of mature bovine adrenodoxin in Escherichia coli.

Site-directed mutagenesis was utilized to enable direct expression of the mature form of bovine adrenodoxin cDNA using the pKK223-3 expression vector in Escherichia coli. Expression was under control of the "tac" promoter and resulted in a direct expression of soluble mature bovine adrenodoxin (greater than 15 mg per liter). Chromatographic behavior of recombinant adrenodoxin did not differ from that reported for mature native adrenodoxin. The purified recombinant protein was identical to native mitochondrial adrenodoxin on the basis of molecular weight, NH2 terminal sequencing and immunoreactivity. E. coli lysates were brown in color, and the purified protein possessed a visible absorbance spectra identical to native bovine adrenodoxin consistent with incorporation of a [2Fe-2S] cluster in vivo. Recombinant bovine adrenodoxin was active in cholesterol side-chain cleavage when reconstituted with adrenodoxin reductase and cytochrome P450scc and exhibited kinetics reported for native bovine adrenodoxin. The presence of the adrenodoxin amino terminal presequence does not appear to be essential for correct folding of mature recombinant adrenodoxin in E. coli. This expression system should prove useful for overexpression of adrenodoxin mutants in future structure/function studies. The approach described herein can potentially be used to directly express the mature form of any protein in bacteria.