High-level Expression System Research Articles

Elimination of in vivo cleavage between target protein and intein in the intein-mediated protein purification systems

IMPACT™ is a novel protein purification system developed by New England BioLabs. This system utilizes the inducible self-cleavage activity of a protein splicing element (termed intein) to separate the target protein from the affinity intein–CBD tag without using any proteases. The IMPACT™ system has been widely used as an easy, one-step protein purification procedure. However, IMPACT™ usually produces a lower target protein yield than other protein purification systems, such as the pET system. It was found that the lower yield of the target protein, in many cases, was due to in vivo cleavage between the target protein and the intein during protein expression. Self-cleavage requires the first residue of the intein to be a cysteine with a free sulfhydryl group. In order to eliminate this in vivo auto-cleavage, we designed two single cysteine mutants in the intein protein to allow potential disulfide bond formation with the first cysteine. Therefore, no free sulfhydryl group in the first cysteine of the intein is available during protein expression. Our data indicated that the in vivo auto-cleavage during bacterial expression was completely eliminated with the intein mutation, resulting in a significant enhancement of the protein yield of the IMPACT™ expression system, comparable to the other high-level expression systems.

Functional Consequences of a Mutation in an Expressed Human α-Cardiac Actin at a Site Implicated in Familial Hypertrophic Cardiomyopathy

Point mutations in human alpha-cardiac actin cause familial hypertrophic cardiomyopathy. Functional characterization of these actin mutants has been limited by the lack of a high level expression system for human cardiac actin. Here, wild-type (WT) human alpha-cardiac actin and a mutant E99K actin have been expressed and purified from the baculovirus/insect cell expression system. Glu-99 in subdomain 1 of actin is thought to interact with a positively charged cluster located in the lower 50-kDa domain of the myosin motor domain. Actin-activated ATPase measurements using the expressed actins and beta-cardiac myosin showed that the mutation increased the K(m) for actin 4-fold (4.7 +/- 0.7 mum for WT versus 19.1 +/- 3.0 mum for the mutant), whereas the V(max) values were similar. The mutation slightly decreased the affinity of actin for S1 in the absence of nucleotide, which can partly be accounted for by a slower rate of association. The in vitro motility for the E99K mutant was consistently lower than WT over a range of ionic strengths, which is likely related to the lower average force supported by the mutant actin. The thermal stability of the E99K was comparable to that of WT-actin, implying no folding defects. The lower density of negative charge in subdomain 1 of actin therefore weakens the actomyosin interaction sufficiently to decrease the force and motion generating capacity of E99K actin, thus providing the primary insult that ultimately leads to the disease phenotype.

The synthesis and high‐level expression of a β2‐adrenergic receptor gene in a tetracycline‐inducible stable mammalian cell line

High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta2-adrenergic receptor (beta2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta2-AR/mg of membrane protein, as measured by saturation binding assay using the beta2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta2-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 microg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 microg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta2-AR expression using suspension cultures in a bioreactor should now allow production of enough beta2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.

Improvement of protein production in lactic acid bacteria using 5′-untranslated leader sequence of slpA from Lactobacillus acidophilus

The 5'-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5' stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses alpha-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the alpha-amylase mRNA was proved by measuring alpha-amylase activity in the culture supernatant and the relative expression of alpha-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving alpha-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.

Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris

High-level recombinant expression systems for the production of stable triple-helical human collagens and collagen fragments have been developed in the yeast Pichia pastoris. Collagen fragments are secreted as single-chain polypeptides by the yeast alpha-mating factor pre-pro sequence, but secretion of full-length triple-helical procollagen molecules has not been achieved despite the use of the same secretory signal. We studied here the effects of the secretory signal and the conformation and size of the collagen polypeptide on its secretion in P. pastoris. Unlike the collagen signal sequence, the alpha-mating factor pre-pro sequence led to efficient secretion of single-chain 45 and 9 kDa type I collagen fragments. The efficiency was dependent on the length of the collagen polypeptide, as secretion of single-chain full-length 90 kDa alpha1(I) polypeptides was less efficient than that of the 45 kDa fragment. Furthermore, the conformation of the collagen polypeptides had a marked effect on secretion, as induction of trimerization of the 45 and 9 kDa fragments by either the C propeptide or the small trimerizing domain foldon led to an accumulation of triple-helical molecules inside the cells despite the presence of the alpha-mating factor pre-pro sequence. Our results show that P. pastoris is a suitable host for the development of tailored expression systems aimed at selective production of nonsecreted triple-helical and secreted single-chain collagen fragments of varying lengths for specific purposes.

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

Expression, Crystallization, and Three-dimensional Structure of the Catalytic Domain of Human Plasma Kallikrein

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 A for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 A for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.

Production of active recombinant mitogen-activated protein kinases through transient transfection of 293T cells

Mitogen-activated protein (MAP) kinases are a family of serine/threonine protein kinases that play an important role in a myriad of cellular processes, including cell proliferation, differentiation, and apoptosis. Abnormal activation of MAP kinases has been shown to participate in a variety of human diseases which include cancer, septic shock, rheumatoid arthritis, diabetes, and cardiovascular diseases. Active MAP kinase enzymes are not only valuable for basic biomedical research but are also critical for the development of pharmacological inhibitors as therapeutic drugs in the treatment of relevant human diseases. MAP kinases produced in a bacterial system are poorly active due to a lack of proper phosphorylation at their characteristic threonine and tyrosine residues. To overcome these limitations, we have developed a mammalian expression system for high level expression and one-step purification of enzymatically MAP kinases. We cloned JNK1, p38, and p38-regulated MAP kinase-activated protein kinase-2 into the mammalian expression vector pEBG, and expressed these protein kinases as glutathione S-transferase fusion proteins in human embryonic kidney 293T cells through transient transfection. The protein kinases were activated in vivo through treating the transfected cells with sodium arsenite and affinity-purified using glutathione–Sepharose beads. The enzymatic activities of these protein kinases were demonstrated by Western blot analysis and in vitro kinase assays. Our results indicate that this system is an extremely powerful tool for generating valuable reagents, and could be very valuable for proteomic studies.

Tolerance of the Rieske-type [2Fe-2S] cluster in recombinant ferredoxin BphA3 from Pseudomonas sp. KKS102 to histidine ligand mutations.

BphA3 from Pseudomonas sp. KKS102 is a Rieske-type [2Fe-2S] ferredoxin that transfers electrons from an NADH-dependent oxidoreductase, BphA4, to a biphenyl dioxygenase complex. A high-level expression and purification system for the recombinant BphA3 in Escherichia coli was constructed. Two histidine ligands of the Rieske-type cluster in BphA3, were each replaced with serine, cysteine, asparagine and tyrosine. The single mutants, in which either His44 or His65 was replaced with a cysteine residue (CH and HC mutants respectively), and the double mutant, in which both histidine residues were replaced with cysteine residue (CC mutant), accumulated to high levels in the E. coli cells, while the other single mutants did not. The purified WT (wild-type) protein showed characteristic near-UV and visible absorption and CD spectra of Rieske-type clusters. The X-ray absorption spectra were suggestive of the existence of [2Fe-2S] clusters, with one histidine and three cysteine ligands in the CH and HC mutants, and an [2Fe-2S] cluster with four cysteine ligands in the CC mutant. The BphA4-dependent cytochrome c reductase activities of the mutants were less than 0.3% of that of the WT protein. The redox potential of the WT protein determined by cyclic voltammetry was -180+/-5 mV compared with the standard hydrogen electrode, and that of the CH mutant was approx. 175 mV lower. The changes in the near-UV and visible absorption spectra of the mutants showed that the reduced iron-sulphur clusters in the mutants were unstable. His44 and His65 in BphA3 can be replaced with cysteine residues, but are required for the stabilization of the reduced form of the cluster.

Polychlorinated biphenyl rhizoremediation by Pseudomonas fluorescens F113 derivatives, using a Sinorhizobium meliloti nod system to drive bph gene expression.

Rhizoremediation of organic chemicals requires high-level expression of biodegradation genes in bacterial strains that are excellent rhizosphere colonizers. Pseudomonas fluorescens F113 is a biocontrol strain that was shown to be an excellent colonizer of numerous plant rhizospheres, including alfalfa. Although a derivative of F113 expressing polychlorinated biphenyl (PCB) biodegradation genes (F113pcb) has been reported previously, this strain shows a low level of bph gene expression, limiting its rhizoremediation potential. Here, a high-level expression system was designed from rhizobial nod gene regulatory relays. Nod promoters were tested in strain F113 by using beta-galactosidase transcriptional fusions. This analysis showed that nodbox 4 from Sinorhizobium meliloti has a high level of expression in F113 that is dependent on an intact nodD1 gene. A transcriptional fusion of a nodbox cassette containing the nodD1 gene and nodbox 4 fused to a gfp gene was expressed in the alfalfa rhizosphere. The bph operon from Burkholderia sp. strain LB400 was cloned under the control of the nodbox cassette and was inserted as a single copy into the genome of F113, generating strain F113L::1180. This new genetically modified strain has a high level of BphC activity and grows on biphenyl as a sole carbon and energy source at a growth rate that is more than three times higher than that of F113pcb. Degradation of PCBs 3, 4, 5, 17, and 25 was also much faster in F113L::1180 than in F113pcb. Finally, the modified strain cometabolized PCB congeners present in Delor103 better than strain LB400, the donor of the bph genes used.

Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system

The cannabinergic system is present in a variety of organs and tissues that perform a wide range of essential physiologic functions making it an inherently important therapeutic target for drug discovery. In order to augment our knowledge regarding the interactions between cannabinoid receptors (CBs) and their ligands, efficient and effective tools are essential for robust expression and purification of these membrane-bound proteins. In this report, we describe a suitable method for purification of the human cannabinoid receptor 2 (CB2) to a qualitative and quantitative level sufficient for mass spectral analysis. We utilized a baculovirus expression system, incorporating several epitope tags to facilitate purification and to ameliorate the effect the tags have on CB2 expression and function. Expressed protein encoded by a carboxy (C)-terminal His-tagged CB2 construct displayed a B(max) value of 9.3 pmol/mg with a K(D) of 7.30 nM using [3(H)]CP-55(940), a standard cannabinoid radioligand, and was selected for subsequent purification experiments. Western blot analysis of purified membrane protein yielded several forms of CB2, the most abundant being a 41 kDa peptide. A second protein species was observed with an apparent molecular weight of 46 kDa representing a glycosylated form of CB2. In addition, a CB2 homodimer was also identified. The purified receptor was subjected to mass spectroscopic analysis to confirm its identity and purity. Mass spectra corresponding to the intracellular, extracellular and transmembrane domains were obtained. These experiments exemplify the importance of high-level expression systems when developing membrane-bound protein purification strategies. This work will aid in the identification of receptor-ligand binding sites, the characterization of molecular features involved in receptor activation, and the elucidation of the CB2 receptor tertiary structure.

Structure and mechanism of monoamine oxidase.

Monoamine oxidases A and B (MAO A and MAO B) are mitochondrial outer membrane-bound flavoproteins that catalyze the oxidative deamination of neurotransmitters and biogenic amines. A number of mechanism-based inhibitors (MAOI's) have been developed for clinical use as antidepressants and as neuroprotective drugs. To facilitate the development of more effective and specific inhibitors, a detailed understanding of the structures and catalytic mechanisms of these enzymes is required. The recent development of high level expression systems for producing recombinant human liver MAO A and MAO B in Pichia pastoris has facilitated the determination of the three dimensional crystal structures of MAO B (up to 1.7 angstroms resolution) in complex with different reversible (isatin, 1,4-diphenyl-2-butene) and irreversible inhibitors (pargyline, N-(2-aminoethyl)-p-chlorobenzamide, and trans-2-phenylcyclopropylamine). The binding of substrates or inhibitors to MAO B involves an initial negotiation of a protein loop occurring near the surface of the membrane and two hydrophobic cavities; an "entrance" cavity and an "active site" cavity. These two cavities can either be separate or in a fused state depending on the conformation of the Ile199 side chain, which appears to function as a gate. The amine function of the bound substrate approaches the re face of the bent and "puckered" covalent FAD through an "aromatic cage" formed by two tyrosine residues that are perpendicular to the plane of the flavin ring. No amino acid residues that could function as acids or bases are found near the catalytic site. The existing structural data on MAO B support previous QSAR results and are also supportive of a proposed polar nucleophilic mechanism for MAO A and B catalysis rather than the alternatively proposed single electron transfer mechanism.

The Interferon-α Genes from Three Chicken Lines and Its Effects on H9N2 Influenza Viruses

The interferon-α genes from three chicken lines were cloned by a direct PCR technique, and the effects of recombinant protein expressed in a prokaryotic system on highly pathogenic H9N2 influenza viruses were investigated. The cloned ChIFN-α gene encoded a protein of 193 amino acids with a signal sequence of 31 amino acids and mature peptides of 162 amino acids. Comparison of ChIFN-α sequences, detected six amino acids substitutions at positions 50, 58, 65, 81, 181, and 183. Homology analysis indicated that ChIFN-α genes could be subdivided into two lineages, SH-ChIFN-α and WJ-ChIFN-α. In addition, both SH-ChIFN-α and WJ-ChIFN-α were expressed with the N-terminal 6 consecutive histidine residues in a high-level prokaryotic expression system. Recombinant chicken interferon-α (rChIFN-α) protein has anti-VSV activity of more than 1 × 108 U/mg. Moreover, High concentration (10,000 U) of rSH-ChIFN-α resulted in over 40% inhibition of the H9N2 virus infection in chicken embryos (Ovo), and 100% inhibition from one day-old to five day-old chickens (Vivo). The results suggested that rChIFN-α is a potential agent against many Chicken viral strains.

Influence of expressed TRAIL on biophysical properties of the human leukemic cell line Jurkat

The cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) was cloned into RevTet-On, a Tet-regulated and high-level gene expression system. The gene expression system was constructed in a human leukemic cell line: Jurkat. By using RevTet-On TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the changes of cellular apoptosis before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the cellular apoptosis ratio was largely dependent on the trail gene expression level. Moreover, it was found that the apoptosis-inducing TRAIL could cause significant changes in the biophysical properties of Jurkat cells. The cell surface charge density decreased, the membrane fluidity declined, the elastic coefficients K1 increased, and the proportion of a-helix in membrane protein secondary structure decreased. Thus, the apoptosis-inducing TRAIL gene caused significant changes on the biomechanic properties of Jurkat cells.

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.

Expression, Purification, and Characterization of Humanized Anti-HBs Fab Fragment

Anti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV. Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris. This was achieved by co-integration of the genes encoding the heavy and light chains both under the genome of the yeast cells. The Fab fragment was efficiently secreted into medium at a concentration of 50 mg/liter. The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of approximately 50 kDa under nonreducing conditions and two bands of approximately 28 kDa under reducing conditions. The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography. The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1 mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg). The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.

High-level expression of recombinant rabbit cytochrome P450 2E1 in Escherichia coli C41 and its purification

Cytochrome P450 2E1 (CYP2E1) is of great interest because of its important role in the oxidation of numerous drugs and carcinogens. The yields of CYP2E1 obtained by the traditional recombinant expression systems have been relatively poor. We report here the development of a system for high-level expression of rabbit CYP2E1 in Escherichia coli strain C41 (DE3). Expression of the membrane-bound CYP2E1 by the pLW01-P450 expression plasmid, which utilizes a T7 promoter, is markedly improved by employing E. coli strain C41 (DE3). The pLW01/2E1 expression plasmid was successfully constructed and high-level expression of CYP2E1 was achieved, which ranged between 900 and 1400 nmol (liter culture) −1. This yield was 9–14-fold higher than other reports of CYP2E1 expression in other E. coli strains. This system provides a highly efficient tool for expressing CYP2E1. An improved purification procedure for the expressed CYP2E1 involving chromatography on diethylaminoethyl cellulose (DE52), Reactive Red–agarose (type 1000-CL), and hydroxyapatite is also reported. This procedure allowed recovery of 45% of the expressed protein and CYP2E1 with a specific content of 14 nmol/mg protein, which showed a single band on a polyacrylamide gel stained with Coomassie brilliant blue.

High-level expression of porcine liver cytochrome P-450 reductase catalytic domain in Escherichia coli by modulating the predicted local secondary structure of mRNA.

A direct expression system for the solubilized catalytic domains of NADPH-cytochrome P-450 reductase (sCPR) from rat (RsCPR) and porcine (PsCPR) in Escherichia coli cells was constructed using the expression plasmid pCWori(+). PsCPR was minimally expressed, whereas RsCPR was highly expressed. Replacement of the nucleotides encoding Thr(60)Ser(61)Ser(62) in PsCPR with those for Ala(60)Pro(61)Pro(62) in RsCPR markedly increased the expression level of the protein. The local secondary structures of the mRNAs, which were predicted with the prediction program GeneBee (http://www.genebee.msu.su), suggested that the intramolecular double strand between the ribosome binding site (RBS) and the Thr(60)Ser(61)Ser(62) codons in PsCPR, and/or the base-pairing at the initiation codon of the mRNAs significantly affected protein expression. Silent mutations were systematically introduced into the codons for Thr(58) and Thr(60)Ser(61) in PsCPR to modulate the local secondary structure of the mRNA. The expression level of the silently mutated PsCPR suggests that the expression level of PsCPR depends on the stability of the local structure at the RBS in the mRNA. A high-level expression system for wild-type PsCPR was constructed by introducing silent mutations at the codons for Thr(60)Ser(61) in PsCPR. The purified PsCPR showed the characteristic absorption spectral changes of sCPR after reduction with NADPH. The yield of purified PsCPR from 1 liter of culture fluid was 45.8 mg. These results substantiate that the introduction of silent mutations in the section of the gene encoding the N-terminal region of the protein based on the predicted local secondary structure of the mRNA at the RBS is a useful approach to control and increase the expression level of heterologous proteins in E. coli cells.

Cloning and expression of the superoxide dismutase gene from the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F).

We identified the SOD gene in the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F) and constructed a high-level expression system in Escherichia coli. A 2.6-kbp DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with EcoRI and SmaI contained the SOD gene and part of another open reading frame. The amino acid sequence deduced from the SOD gene, which was composed of 238 amino acid residues, showed high homogeneity with iron-containing SOD (Fe-SOD) and predicted that the amino terminus of this protein would carry an export signal peptide. We produced the precursor form of SOD (PSOD) and the mature form of SOD (MSOD), which lacked the putative signal peptide. In E. coli, PSOD was present in insoluble inclusion bodies, and its putative signal peptide was not cleaved. In contrast, MSOD contained one iron per mononer and formed a dimer, which exhibited an SOD activity of 850 U/mg. Furthermore, D. vulgaris soluble extract showed a band of SOD activity on native polyacrylamide gel that migrated to the same point as MSOD. The intracellular localization of SOD and its role in D. vulgaris are also discussed.

A thermostable laccase from Streptomyces lavendulae REN-7: purification, characterization, nucleotide sequence, and expression.

We found a polyphenoloxidase (PPO) in the cell extract of Streptomyces lavendulae REN-7. About 0.8 mg of purified PPO was obtained from 200 g of the mycelia with a yield of 9.0%. REN-7-PPO showed broad substrate specificity toward various aromatic compounds. Moreover, this enzyme was capable of oxidation of syringaldazine, which is a specific substrate for laccase. Interestingly, REN-7-PPO retained its original activity after 20 min of incubation at even 70 degrees C. The gene encoding the PPO was cloned. Four copper-binding sites characteristics of laccases were contained in the deduced amino acid sequence. We constructed a high-level expression system of this gene in Escherichia coli. The properties of the recombinant enzyme were identical that of wild-type. In conclusion, this PPO is a thermostable laccase.