T7 RNA Polymerase Expression Research Articles

Expression of the mau gene cluster of Paracoccus denitrificans is controlled by MauR and a second transcription regulator.

The mau gene cluster of Paracoccus denitrificans constitutes 11 genes (10 are located in the transcriptional order mauFBEDACJGMN; the 11th, mauR, is located upstream and divergently transcribed from these genes) that encode a functional methylamine-oxidizing electron transport branch. The mauR gene encodes a LysR-type transcriptional activator essential for induction of the mau operon. In this study, the characteristics of that process were established. By using lacZ transcriptional fusions integrated into the genome of P. denitrificans, it was found that the expression of the mauR gene during growth on methylamine and/or succinate was not autoregulated, but proceeded at a low and constant level. The mauF promoter activity was shown to be controlled by MauR and a second transcriptional regulator. This activity was very high during growth on methylamine, low on succinate plus methylamine, and absent on succinate alone. MauR was overexpressed in Escherichia coli by using a T7 RNA polymerase expression system. Gel shift assays indicated that MauR binds to a 403 bp DNA fragment spanning the mauR-mauF promoter region. It is concluded from these results that the expression of the structural mau genes is dependent on MauR and its inducer, methylamine, as well as on another transcription factor. Both activators are required for high-level transcription from the mauF promoter. It is hypothesized that the two activators act synergistically to activate transcription: the effects of the two activators are not additive and either one alone activates the mauF promoter rather weakly.

Rescue of rinderpest virus from cloned cDNA.

Rinderpest virus is a morbillivirus and is the causative agent of a widespread and important disease of cattle. The viral genome is a single strand of RNA in the negative sense. We have constructed plasmids containing cDNA copies of the 5' and 3' termini of the virus separated by a reporter gene and have shown that antigenome-sense RNA transcripts of these model genomes can be replicated, transcribed, and packaged by helper virus, both rinderpest virus and the related measles virus. Further, these genome analogs can be replicated and transcribed by viral proteins expressed from cDNA clones by using a recombinant vaccinia virus expressing T7 RNA polymerase (MVA-T7). Using this latter system, we have rescued live rinderpest virus from a full-length cDNA copy of the genome of the RBOK vaccine strain. The recombinant virus appears to grow in tissue culture identically to the original virus.

Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.

An artificial regulatory circuit for stable expression of DNA-binding proteins in a T7 expression system

We had earlier overproduced the transcription activator protein C of bacteriophage Mu in a phage-T7 expression system. Although we achieved a high level of overproduction, the expression was not consistent. This could be due to the leaky expression of T7 RNA polymerase in the uninduced state. Introduction of pLysS, a plasmid encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase, resulted in consistent, but extremely low production of the C protein. To overcome this problem, we have devised an artificial regulatory circuit to obtain stabilised, consistent overproduction of C protein. The C-binding site was cloned downstream from the transcription start point of T7 lys. Upon induction, the C protein produced binds to its site with a very high affinity, possibly acting as a transcriptional roadblock for lys. This would overcome the inhibitory effect of T7 lysozyme on T7 RNA polymerase.

Construction and oral immunogenicity of a Salmonella typhimurium strain expressing a streptococcal adhesin linked to the [formula omitted] subunits of cholera toxin

A mafor adhesin from the oral pathogen Streptococcus mutans has been shown to be mucosally immunogenic upon genetic fusion with the cholera toxin A2 B subunits. To take advantage of the ability of Salmonella typhimurium to deliver cloned antigens to the mucosal inductive sites that would obviate the need for antigen purification, we expressed this chimeric construct in an attenuated S. typhimurium strain under the control of bacteriophage T7 transcription. Residual expression of the temperature-regulated T7 RNA polymerase at 30°C allowed production of the chimeric protein at 2–3% of the total soluble protein, but it was increased five to six times following induction at 37°C. Oral administration of a single dose of 10 9 recombinant Salmonella to mice resulted in serum IgG and salivary IgA antibody responses to Salmonella, cholera toxin, and the streptococcal adhesin, which were generally enhanced after a booster immunization.

Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase.

A large variety of different RNA species that are replicated by DNA-dependent RNA polymerase from bacteriophage T7 have been generated by incubating high concentrations of this enzyme with substrate for extended time periods. The products differed from sample to sample in molecular weight and sequence, their chain lengths ranging from 60 to 120. The mechanism of autocatalytic amplification of RNA by T7 RNA polymerase proved to be analogous to that observed with viral RNA-dependent RNA polymerases (replicases): only single-stranded templates are accepted and complementary replica strands are synthesized. With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template. The plus strands, present at 90% of the replicating RNA species, were found to have GG residues at both termini. Consensus sequences were not found among the sequences of the replicating RNA species. The secondary structures of all species sequenced turned out to be hairpins. The RNA species were specifically replicated by T7 RNA polymerase; they were not accepted as templates by the RNA polymerases from Escherichia coli or bacteriophage SP6 or by Qbeta replicase; T3 RNA polymerase was partially active. Template-free production of RNA was completely suppressed by addition of DNA to the incubation mixture. When both DNA and RNA templates were present, transcription and replication competed, but T7 RNA polymerase preferred DNA as a template. No replicating RNA species were detected in vivo in cells expressing T7 RNA polymerase.

Expression and Spectroscopic Characterization of the Hydrogenosomal [2Fe-2S] Ferredoxin from the Protozoan Trichomonas vaginalis

The heterologous expression and spectroscopic characterization of the [2Fe-2S] ferredoxin from the sexually transmitted human parasite Trichomonas vaginalis is described. Using oligonucleotide primers based on the deduced DNA sequence, the gene encoding the ferredoxin was amplified by polymerase chain reaction and cloned into a T7 RNA polymerase expression vector. Expression of the gene in Escherichia coli host HMS174(DE3) resulted in the high level production of the protein with the correctly assembled iron-sulfur cluster. The absorption, circular dichroism, resonance Raman, and EPR spectra of the recombinant protein revealed many differences from those of other [2Fe-2S] ferredoxins. The redox potential of the protein (-347 mV versus normal hydrogen electrode) was also determined. Whereas the amino acid sequence of T. vaginalis ferredoxin showed greatest homology to the [2Fe-2S] ferredoxins found in bacteria and vertebrate mitochondria which function in cytochrome P450 oxidation pathways, the spectroscopic properties showed substantial dissimilarity. Differences in the biophysical properties and function of T. vaginalis ferredoxin are proposed to result from the characteristic amino acid sequence of the parasite protein near the cysteine residues that ligate the valence-localized Fe(III) site of the reduced cluster.

Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox virus.

The bacteriophage T7 RNA polymerase gene was integrated into the fowlpox virus genome under the control of the vaccinia virus early/late promoter, P7.5. The recombinant fowlpox virus, fpEFLT7pol, stably expressed T7 RNA polymerase in avian and mammalian cells, allowing transient expression of transfected genes under the control of the T7 promoter. The recombinant fowlpox virus expressing T7 RNA polymerase offers an alternative to the widely used vaccinia virus vTF7-3, or the recently developed modified vaccinia virus Ankara (MVA) T7 RNA polymerase recombinant, a highly attenuated strain with restricted host-range. Recombinant fowlpox viruses have the advantage that as no infectious virus are produced from mammalian cells they do not have to be used under stringent microbiological safety conditions.

A replication-deficient adenovirus enhances liposome-mediated nucleic acid transfer into a stable cell line expressing T7 RNA polymerase.

Liposome-mediated transfer of nucleic acids into a cell line expressing bacteriophage T7 RNA polymerase was enhanced by addition of a replication-deficient adenovirus (Ad5-259A) to transfection mixtures. Increasing quantities of Ad5-259A resulted in a dose-related (up to 30-fold) enhancement of reporter gene activity expressed in BT7-H cells transfected with plasmid DNA containing the reporter sequence fused to the internal ribosome entry site of encephalomyocarditis virus. Similarly, Ad5-259A enhanced reporter gene expression 7-fold following transfection of DNA containing the reporter sequence under transcriptional control of the Rous sarcoma virus long terminal repeat. Addition of Ad5-295A to transfection mixtures increased the proportion of cells staining positively for reporter gene activity, from 2 to 25% when the reporter was expressed via the T7 polymerase and from 20 to 50% when the reporter was under the control of a eucaryotic promoter. Thus, Ad5-259A enhanced reporter protein activities expressed by cytoplasmic T7-directed transcription and cap-independent initiation of translation, or nuclear transcription and cap-dependent translation. Transfection enhancement was blocked by neutralizing antibody to Ad5, and is most likely related to the endosome-disrupting activities of the virus. Adenovirus enhancement of liposome-mediated transfection provides a useful method for efficient nucleic acid transfer into eucaryotic cells.

Transcripts containing a small anti-HIV hammerhead ribozyme that are active in the cell cytoplasm but inactive in vitro as free RNAs

In order to study the activity of a hammerhead ribozyme in a cytoplasmic environment, HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase were cotransfected with plasmids expressing the ribozyme and its target RNA (nucleotides (nt) +1 to +692 of HIV-1 RNA) under the control of a T7 promoter. Two ribozyme-containing plasmids were designed to express RNAs of respectively 181 nt (Rz181) and 132 nt (Rz132). The sequence of each of these RNAs contained a 35 nt hammerhead ribozyme which is known to cleave its minimal 14-mer RNA substrate efficiently in vitro at a site corresponding to position +115 of the HIV-1 RNA. Control transfections were carried out with the parental plasmid pET3, which expressed a 134 nt RNA lacking the ribozyme sequence, and also with a plasmid expressing a 181 nt RNA (Rz181M) containing a single mutation known to inactivate the in vitro cleavage activity of the ribozyme. As detected by RT-PCR, the amount of target RNA was reproducibly reduced at a ribozyme/target ratio higher than 50 with Rz181 and Rz132 whereas it remained unaffected with Rz181M, thus eliminating the possibility of antisense inhibition. Rz132 proved to be more efficient than Rz181. Competitive RT-PCR indicated that, at ribozyme/target ratio of 300, the amount of residual target RNA was reduced by approximately 85% in the presence of Rz181. In contrast to these in vivo effects, Rz181 and Rz132 obtained by in vitro transcription were inactive against the minimal 14 mer (or longer) substrate under a variety of conditions. In conclusion, although in vitro studies of ribozymes are essential to learn their catalytic mechanism, they cannot be used to predict the efficiency of RNAs containing a ribozyme sequence when it is expressed in cells.

Cloning and expression of the α-amylase gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1, and characterization of the enzyme

A hyperthermophilic archaeon, Pyrococcus sp. KOD1, produces extracellular thermostable α-amylase (ApkA). The apkA gene was cloned and expressed in Escherichia coli. Nucleotide sequence analysis showed that the apkA gene is composed of 1,383 bp (461 amino acid residues) and contains a signal sequence (26 N-terminal amino acid residues). The molecular weight of the mature enzyme was estimated to be 49,456 Da with 435 amino acid residues. The deduced amino acid sequence of the mature enzyme was compared with those of α-amylase and other starch-degrading enzymes from a variety of organisms. The overall homology to other amylases was not high (below 40%); however, high homology was found in the four conserved regions of the α-amylase family. The cloned apkA was overexpressed using the T7 RNA polymerase expression system. The enzyme was purified by ammonium sulfate fractionation, heat treatment, anion-exchange chromatography, and hydrophobic interaction chromatography. The optimum temperature and pH for the enzyme activity were found to be 90°C and pH 6.5, and the enzyme was considerably stable even after heating at 90°C for 60 min. The thermostability of the enzyme was enhanced in the presence of 2 mM Ca2+. ApkA hydrolyzed soluble starch and glycogen to form maltose and maltotriose as the major products, and very weakly hydrolyzed pullulan to form panose or isopanose, suggesting cleavage at the α-1,4-linkage of pullulan. The steady-state kinetic constants were determined for various substrates, and ApkA was found to show high affinity to highly branched polysaccharides such as glycogen.

Cloning, sequencing, overexpression in Escherichia coli, and inactivation of the valine dehydrogenase gene in the polyether antibiotic producer Streptomyces cinnamonensis

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide antibiotic biosynthesis. To determine for the first time the importance of valine dehydrogenase (vdh) in polyether antibiotic biosynthesis, the vdh gene from Streptomyces cinnamonensis has been cloned and sequenced. The enzyme (M r 37 718 Da) has been produced in large amounts in an active form in the E. coli cytoplasm using a T7 RNA-polymerase expression system. Upon inactivation of the gene in S. cinnamonensis by a double-crossover mechanism, a hyg::vdh mutant was isolated that was devoid of vdh activity. Upon growth in chemically defined media, as well as a complex medium optimised for monensin production, the mutant and wild-type grew equally well and reached the same levels of monensin production. In both strains a valine transaminase activity could be detected that provides an alternative route for converting valine into 2-oxoisovaleric acid. The results show that vdh is not essential for normal growth of S. cinnamonensis, and its inactivation does not significantly affect normal levels of monensin production in this strain.

A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus.

We have recovered infectious Sendai virus (SeV) from full-length cDNA (FL-3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL-3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5' nontranslated region of the NP gene, creating a new, out-of-frame, 5' proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL-3 and pGEM-NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL-3 with that of pGEM-NP. When a fifth plasmid containing a new genomic 3' end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3' end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy-back nondefective virus was generated which interferes with wild-type virus replication.

Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system

cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleave this molecule to 1AB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant VVs constitutively expressing these cassettes were unsuccessful. However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated. Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins. Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed. Electron microscopy revealed empty capsid-like particles with diameters of about 30 nm. Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles. Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins. However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles.

Rescue of measles viruses from cloned DNA.

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.

Overexpression inEscherichia coli,Folding, Purification, and Characterization of the First Three Short Consensus Repeat Modules of Human Complement Receptor Type 1

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system inEscherichia coliwas used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP–Sepharose. The oligomer was folded by one-step dilution in 20 mMethanolamine/1 mMEDTA supplemented with 1 mMGSH/0.5 mMGSSG. The folded material was processed to a concentrated (>20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1–3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 × 110 Å. SCR(1–3) has an unusual CD spectrum exhibiting a broad maximum at 220–230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.

Purification, crystallization, and preliminary X-ray analysis of Bacillus subtilis ferrochelatase.

Bacillus subtilis ferrochelatase (EC 4.99.1.1), the final enzyme in protoheme IX biosynthesis, was produced with an inducible T7 RNA polymerase expression system in Escherichia coli and purified from the soluble cell fraction. It was crystallized from polyethylene glycol solution using the microseeding technique. The crystals diffract to a minimum Bragg spacing of 2.1 A. The space group is P4(2) with unit cell dimensions a = b = 50.2 A, c = 120.1 A.

High-Yield Expression, Refolding, and Purification of Penicillin-Binding Protein 2a from Methicillin-Resistant Staphylococcus aureus Strain 27R

The mecA-27R gene, which encodes PBP2a from methicillin-resistant Staphylococcus aureus strain 27R, was modified to remove the putative N-terminal membrane-spanning region, cloned into the T7 RNA polymerase expression vector pET11d, and used to transform Escherichia coli strain BL21(DE3). The majority of PBP2a was expressed in the form of inclusion bodies, which were extracted, denatured, and refolded, The protein was then purified by anion-exchange and size-exclusion chromatography, A B-liter culture of induced E. coli provided 37 mg of purified PBP2a which was greater than 99% pure, finding affinities for [3H]benzylpenicillin, imipenem, and L-695,256 (a β-lactam with high affinity for PBP2a) were shown to be comparable to PEP2a found in membrane preparations of S. aureus strain 27R, A direct binding assay, using 14C-labeled L-695,256 was developed and used to show stoichiometric binding to the refolded, soluble PBP2a, In addition, electrospray mass spectrometry showed that 100% of the refolded PBP2a was covalently bound to the β-lactam in a stoichiometric fashion. Finally, two mutations of the putative active-site serine showed the predicted loss of covalent binding of the β-lactam to the PBP2a, demonstrating the high specificity of the soluble binding assay.

RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA

Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a approximately 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was "rescued," as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P.L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3' end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.

Molecular Characterization of Monodehydroascorbate Radical Reductase from Cucumber Highly Expressed in Escherichia coli

Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is the first enzyme to be identified whose substrate is an organic radical and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA has been cloned from cucumber seedlings (Sano, S., and Asada, K. (1994) Plant Cell Physiol. 35, 425-437), and a plasmid was constructed in the present study that allowed a high level expression in Escherichia coli of the cDNA-encoding MDA reductase using the T7 RNA polymerase expression system. The recombinant MDA reductase was purified to a crystalline state, with a yield of over 20 mg/liter of culture, and it exhibited spectroscopic properties of the FAD similar to those of the enzyme purified from cucumber fruits during redox reactions with NADH and MDA. The red semiquinone of the FAD of MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the reduced enzyme to MDA. The specificity of electron acceptors of the recombinant enzyme appeared to be similar to that of MDA reductase, even though the amino acid sequence encoded by the cDNA was somewhat different from that of the enzyme purified from cucumber fruits. The Km values for NADH and NADPH of the recombinant enzyme indicated a high affinity of the enzyme for NADH. The reaction catalyzed by the enzyme did not exhibit saturation kinetics with MDA up to 3 microM. A second order rate constant for the reduction of the enzyme-FAD with NADH was 1.25 x 10(8) M-1 s-1, as determined by a stopped-flow method, and its value decreased with increases in ionic strength, an indication of the enhanced electrostatic guidance of NADH to the enzyme-FAD.