Coli BL21 CodonPlus Research Articles

Technical Data of Heterologous Expression and Purification of SARS-CoV-2 Proteases Using Escherichia coli System

The SARS-CoV-2 coronavirus expresses two essential proteases: firstly, the 3Chymotrypsin-like protease (3CLpro) or main protease (Mpro), and secondly, the papain-like protease (PLpro), both of which are considered as viable drug targets for the inhibition of viral replication. In order to perform drug discovery assays for SARS-CoV-2, it is imperative that efficient methods are established for the production and purification of 3CLpro and PLpro of SARS-CoV-2, designated as 3CLpro-CoV2 and PLpro-CoV2, respectively. This article expands the data collected in the attempts to express SARS-CoV-2 proteases under different conditions and purify them under single-step chromatography. Data showed that the use of E. coli BL21(DE3) strain was sufficient to express 3CLpro-CoV2 in a fully soluble form. Nevertheless, the single affinity chromatography step was only applicable for 3CLpro-CoV2 expressed at 18 °C, with a yield and purification fold of 92% and 49, respectively. Meanwhile, PLpro-CoV2 was successfully expressed in a fully soluble form in either BL21(DE3) or BL21-CodonPlus(DE3) strains. In contrast, the single affinity chromatography step was only applicable for PLpro-CoV2 expressed using E. coli BL21-CodonPlus(DE3) at 18 or 37 °C, with a yield and purification fold of 86% (18 °C) or 83.36% (37 °C) and 112 (18 °C) or 71 (37 °C), respectively. The findings provide a guide for optimizing the production of SARS-CoV-2 proteases of E. coli host cells.

Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells

Streptavidin is a homotetrameric protein produced by Streptomyces avidinii, each subunit of which binds biotin (vitamin H), forming a stable complex (Kd = 10-15 М). Streptavidin-biotin coreaction is widely used in analytical systems, for targeted delivery of compounds, for affinity purification, etc. The aim of this study was to develop a rational technique to produce functionally active recombinant streptavidin. Recombinant Escherichia coli strains producing minimal core and full-sized streptavidin variants were obtained. The E. coli BL21 Codon Plus (DE3) RIPL, as host cells, and the pET19b plasmid carrying gene of minimally-sized core (miniSAV) or full-sized (SAV) streptavidin were used. Synthesis of miniSAV results in its localization as insoluble inclusion bodies. Denatured miniSAV yield was 130 mg per liter of E. coli c ulture. T he r enaturation g ives o nly 10- 15 % of the functionally active protein. Full-sized streptavidin localizes in the cytoplasm in a soluble state, but its toxicity causes low yield of the protein (10-13 mg per liter of the culture). The induction of SAV synthesis at the end of the logarithmic stage of cell growth was found to increase the yield of SAV approximately 2-fold. The yield of functionally active protein was 30 mg per liter culture. SAV was produced practically in individual state after affine chromatography on 2-iminobiotin agarose. One molecule of full-sized streptavidin bound 3.9 biotin molecules as was shown by colorimetric analysis using HABA (4-hydroxyazobenzene-2-carboxylic acid). Both streptavidins form sandwichtype complexes with biotinylated molecules in solid-phase microassay conditions. E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV strain was stable during storage with 20 % glycerol at -70 °С, which was shown by repeated two-year reseeding. The streptavidin producing strain (E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV) is deposited in the Collection for extremophile microorganisms and type cultures (Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk), No. 3505. The method for producing functionally active recombinant streptavidin developed in this study ensures its availability for biotechnological research

Cloning and Heterologous Expression of Hepatitis B Virus Pre-Surface Antigen 1

Hepatitis B causes major deathly infections in developing countries. The surface antigen preS1, hepatocytes attachment site, plays a crucial role in development and progression of disease. The present study was, therefore, an effort to develop an efficient expression system for PreS1 gene in Esherishia coli. PreS1 gene was amplified and cloned in pTZ57R/T vector. Following confirmation through restriction enzyme digestion and sequencing, it was subcloned in pET22b(+). E. coli BL21 (DE3) CodonPlus cells were transformed with the recombinant plasmid. PreS1 gene was induced using IPTG, the protein was expressed and quantified with Bradford assay and analyzed on SDS-PAGE. Enzyme Linked Immunosorbent Assay and Western Blot analysis were performed for protein integrity and conformation.

Optimization of Conditions for Maximal Production of Recombinant Thermostable Cellulase from Thermotoga naphthophila using E. coli BL21-CodonPlus (DE3) as Expression Host

Optimization of Conditions for Maximal Production of Recombinant Thermostable Cellulase from Thermotoga naphthophila using E. coli BL21-CodonPlus (DE3) as Expression Host

Expression and evaluation of recombinant P32 protein based ELISA for sero-diagnostic potential of capripox in sheep and goats

The study is aimed to develop and evaluate a recombinant P32 protein based ELISA for sero-monitoring and sero-surveillance using known and random/suspected serum samples for capripox infections from sheep and goats. Truncated P32 gene of goatpox virus (with an ORF of 750 bp) was expressed in E. coli BL-21 CodonPlus (DE3)-RIPL cells using pET32a vector and characterized by SDS-PAGE analysis and confirmed by western blotting as 48 kDa polyhistidine-tagged fusion protein. The protein was purified under denaturing conditions using 8M urea and characterized by SDS-PAGE and immunoblotting. The purified protein was used for optimizing ELISA in a chequerboard titration method using anti-GTPV serum as known positive. The optimized conditions were found to be 300 ng of protein/well, 1:10 dilution of antibody, 1:10000 dilution of rabbit anti-goat/sheep conjugate with 3% skim milk powder and 2% gelatin in phosphate buffer saline containing tween-20 as blocking buffer. The expressed protein was specific only for goatpox virus and sheeppox virus but did not react with related viruses of sheep and goats namely orf virus, peste de petits ruminants virus, bluetongue virus and foot and mouth disease virus. The optimized ELISA was evaluated using pre-vaccinated, post-vaccinated and also post-challenge sera. The assay was found to have a diagnostic specificity of 100/98.7% and sensitivity of 97.1/98.1% when compared to whole virus antigen based ELISA/SNT by receiver operating characteristic (ROC) analysis. The optimized ELISA is able to determine the progression of antibody response against GTPV and SPPV following vaccination and challenge in sheep and goats. The rP32 protein based ELISA was evaluated using random field serum samples (n = 1008) suspected for sheeppox and goatpox and it has shown positivity rate as 24.4%. The rP32 protein based ELISA was found to be specific and sensitive for sero-evaluation of sheeppox virus and goatpox virus following vaccination and infection in sheep and goats.

THE EXPRESSION AND PURIFICATION OF OCTA-ARGININE APOPTIN AND ITS ABILITY TO KILL CANCER CELLS

<p><strong>Objective: </strong>In this research, <em>chicken anemia virus</em> apoptin optimized genetically for expression in <em>Escherichia coli</em> and also modified using (His)<sub>6</sub> tag, (Arg)<sub>8</sub> tag, and HlyA tag intended for purification needs, penetration enhancement, and secretion from<em> </em>bacterial host<em> </em>to the growth media.</p><p><strong>Methods</strong><strong>:</strong><strong> </strong>The modified apoptin gene was optimized using an Integrated DNA Technology (IDT). The gene (606 bp) then ordered and synthesized by Eurofins. The apoptin gene was expressed using <em>E. coli</em> BL21 CodonPlus as host, in cultivation temperature of 37 °C, and 25 °C and purified using Ni-NTA agarose beads. The addition of (His)<sub> 6</sub> tag enabled the apoptin to be purified in only one step by using nickel column. The expression and purification data analyzed qualitatively as well as quantitatively using SDS-PAGE. MTT assay was used to identify the antitumor effect of octa arginine-apoptin to two kinds of cancer cells, cervix HeLa cancer cell and colon Widr cancer cell. The viability of cell was analyzed when the cell incubated in the variation concentration protein for 72 h.</p><p><strong>Results: </strong>The constructed apoptin gene were expressed in <em>E. coli </em>successfully. The MTT assay indicated that Octaarginin-Apoptin was able to induce apoptosis of HeLa and Widr cells lines in a dose-dependent manner. The recombinant apoptin without tagging with octa-arginine, have no ability to induce apoptosis of HeLa and Widr cells lines. <strong></strong></p><p class="Abstract"><strong>Conclusion: </strong><em>This octa arginine-apoptin may in the future allow the development of a therapeutic protein that is able to kill cancer cells specifically.</em></p>

Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans.

Production and characterization of egg yolk antibody (IgY) against recombinant VP8-S2 antigen.

Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. VP8 subunit of rotavirus is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Studies showed that immunoglobulin of egg yolk (IgY) from immunized hens has been identified to be a convenient source for specific antibodies for using in immunotherapy and immunodiagnostic to limit the infections. In this study, chimeric VP8-S2 gene was designed using by computational techniques. The chimeric VP8-S2 gene was cloned and sub-cloned into pGH and pET32a (+) vectors. Then, recombinant pET32a-VP8-S2 vector was transferred into E. coli BL21 CodonPlus (DE3). The expressed protein was purified by Ni-NTA chromatography column. Hens were immunized with the purified VP8-S2 protein three times. IgY was purified from egg yolks using polyethylene glycol precipitation method. Activity and specificity of anti-VP8-S2 IgY were detected by dot-blotting, Western-blotting and indirect ELISA. We obtained anti-VP8-S2 IgY by immunizing hens with the recombinant VP8-S2 protein. The anti-VP8-S2 IgY was showed to bind specifically to the chimeric VP8-S2 protein by dot-blotting, Western-blotting analyses and indirect ELISA. The result of this study indicated that such construction can be useful to investigate as candidates for development of detection methods for simultaneous diagnosis of both infections. Specific IgY against the recombinant VP8-S2 could be recommended as a candidate for passive immunization against bovine rotavirus and bovine coronavirus.

Cloning and Characterization of an Armillaria gallica cDNA Encoding Protoilludene Synthase, Which Catalyzes the First Committed Step in the Synthesis of Antimicrobial Melleolides

Melleolides and related fungal sesquiterpenoid aryl esters are antimicrobial and cytotoxic natural products derived from cultures of the Homobasidiomycetes genus Armillaria. The initial step in the biosynthesis of all melleolides involves cyclization of the universal sesquiterpene precursor farnesyl diphosphate to produce protoilludene, a reaction catalyzed by protoilludene synthase. We achieved the partial purification of protoilludene synthase from a mycelial culture of Armillaria gallica and found that 6-protoilludene was its exclusive reaction product. Therefore, a further isomerization reaction is necessary to convert the 6–7 double bond into the 7–8 double bond found in melleolides. We expressed an A. gallica protoilludene synthase cDNA in Escherichia coli, and this also led to the exclusive production of 6-protoilludene. Sequence comparison of the isolated sesquiterpene synthase revealed a distant relationship to other fungal terpene synthases. The isolation of the genomic sequence identified the 6-protoilludene synthase to be present as a single copy gene in the genome of A. gallica, possessing an open reading frame interrupted with eight introns.

Heterologous expression of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Triticum aestivum and Arabidopsis thaliana

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus ® cells and the pRSETB vector was successful for expression of a soluble His 6-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.

Overexpression, Purification, and Characterization of β-Subunit of Group II Chaperonin from Hyperthermophilic Aeropyrum pernix K1

In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin beta-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at 90 degrees C for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43 degrees C and 50 degrees C, respectively. Specifically, the activity of malate dehydrogenase (MDH) at 85 degrees C was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of procarboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

Conformational Transition Associated with E1-E2 Interaction in Small Ubiquitin-like Modifications

Ubiquitin-like modifications regulate nearly every aspect of cellular functions. A key step in these modifications is the recognition of the carrier enzyme (E2) by the activating enzyme (E1). In this study, we have found that a critical E2-binding surface on the E1 of the small ubiquitin-like modifier has unusually high populations in both ordered and disordered states. Upon binding the E2, the disordered state is converted to the ordered state, which resembles the structure of the bound conformation, providing a mechanism to resolve the "Levinthal Paradox" search problem in a folding-upon-binding process. The significance of the folding-unfolding equilibrium is shown by the loss of functions of the mutations that shift the equilibrium to the folded state. This study highlights the importance of conformational flexibility in the molecular recognition event.

Characterization of the cry1Ac17 Gene from an Indigenous Strain of Bacillus thuringiensis subsp. kenyae

An indigenously isolated strain of Bacillus thuringiensis subsp. kenyae exhibited toxicity against lepidopteran as well as dipteran insects. The lepidopteran active cry1Ac protoxin gene coding sequence of 3.5 kb from this strain was cloned into vector pET28a(+). However, it could not be expressed in commonly used Escherichia coli expression hosts, BL21(DE3) and BL21(DE3)pLysS. This gene is classified as cry1Ac17 in the B. thuringiensis toxic nomenclature database. The coding sequence of this gene revealed that it contains about 3% codons, which are not efficiently translated by these expression hosts. Hence, this gene was expressed in a modified expression host, Epicurian coli BL21-Codonplus (DE3)-RIL. The expression of gene yielded a 130-kDa Cry1Ac17 protein. The protein was purified and its toxicity was tested against economically important insect pests, viz., Helicoverpa armigera and Spodoptera litura. LC(50) values obtained against these insects were 0.1 ng/cm(3) and 1231 ng/cm(2), respectively. The higher toxicity of Cry1Ac17 protein, compared to other Cry1Ac proteins, toward these pests demonstrates the potential of this isolate as an important candidate in the integrated resistance management program in India.

ATP-driven Reduction by Dark-operative Protochlorophyllide Oxidoreductase from Chlorobium tepidum Mechanistically Resembles Nitrogenase Catalysis

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1) mg(-1) with K(m) values of 6.1 microm for protochlorophyllide, 13.5 microm for ATP, and 52.7 microm for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second inter-subunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.

Aldehyde oxidoreductase as a biocatalyst: Reductions of vanillic acid

Aldehyde oxidoreductase (carboxylic acid reductase) catalyzes the Mg 2+, ATP and NADPH dependent reduction of carboxylic acids to their corresponding aldehydes. The identification of the gene from Nocardia sp. NRRL 5646 and its expression in E. coli BL21-CodonPlus ®(DE3)-RP/pHAT305 provided an avenue to develop a biocatalyst for reduction of carboxylic acids. In addition to aromatic acids, the recombinant carboxylic acid reductase also accepts several aliphatic mono, di and tri carboxylic acids as substrates. A recently identified Nocardia sp., phosphopantetheinyl transferase gene ( npt) enhanced the activity of carboxylic acid reductase. Coexpression of car and npt in E. coli BL21-CodonPlus ®(DE3)-RP/pPV2.83 resulted in a purified recombinant carboxylic acid reductase with improved specific activity of 2.2 U/mg protein. The utility of the recombinant carboxylic acid reductase as a biocatalyst has been demonstrated using vanillic acid as substrate. E. coli BL21-CodonPlus ®(DE3)-RP/pHAT305 expressing Car reduced 50% of vanillic acid to vanillin in 10 h. E. coli BL21-CodonPlus ®(DE3)-RP/pPV2.83 resting cells expressing Car and Npt reduced 90% of vanillic acid to vanillin in 6 h. Enhanced, in vivo cofactor NADPH regeneration by glucose dehydrogenase ( gdh) was accomplished using E. coli BL21-CodonPlus ®(DE3)-RP/pPV2.85, that carried car, npt, and gdh. Resting cell reactions using E. coli BL21-CodonPlus ®(DE3)-RP/pPV2.85 with in situ product removal by XAD-2 resin efficiently reduced 5 g/L of vanillic and benzoic acids within 2 h.

Functional Implication of the tRNA Genes Encoded in the Chlorella Virus PBCV-l Genome

The prototype Chlorella virus PBCV-l encodes 11 tRNA genes and over 350 protein-encoding genes in its 330 kbp genome. Initial attempts to overexpress the recombinant A189/192R protein, a putative virus attachment protein, in E. coli strain BL21(DE3) SI were unsuccessful, and multiple protein bands were detected on Western blots. However, the full-length A189/192R recombinant protein or fragments derived from it were detected when they were expressed in E. coli BL21 CodonPlus (DE3) RIL, which contains extra tRNAs. Codon usage analysis of the a189/192r gene showed highly biased usage of the AGA and AVA codons compared to genes encoded by E. coli and Chlorella. In addition, there were biases of XXA/U(<TEX>$56\%$</TEX>) and XXG/ C(<TEX>$44\%$</TEX>) in the codons recognized by the viral tRNAs, which correspond to the codon usage bias in the PBCV-1 genome of XXA/U (<TEX>$63\%$</TEX>) over those ending in XXC/G (<TEX>$37\%$</TEX>). Analysis of the codon usage in the major capsid protein and DNA polymerase showed preferential usage of codons that can be recognized by the viral tRNAs. The Asn (AAC) and Lys (AAG) codons whose corresponding tRNA genes are duplicated in the tRNA gene cluster were the most abundant (i.e., preferred) codons in these two proteins. The tRNA genes encoded in the PBCV-l genome seem to play a very important role during the synthesis of viral proteins through supplementing the tRNAs that are frequently used in viral proteins, but are rare in the host cells. In addition, these tRNAs would help the virus to adapt to a wide range of hosts by providing tRNAs that are rare in the host cells.

Identification of PamA as a PII-binding Membrane Protein Important in Nitrogen-related and Sugar-catabolic Gene Expression in Synechocystis sp. PCC 6803

The PII signaling protein plays a pivotal role in the coordination of carbon and nitrogen metabolism in a wide variety of bacteria, Archaea, and plant chloroplasts. By using a yeast two-hybrid screening system, we identified a transmembrane protein, designated PamA (encoded by sll0985), as a PII-binding protein in Synechocystis sp. PCC 6803. The interaction between PII and PamA was confirmed in vitro, and the interaction was inhibited in the presence of ATP and 2-oxoglutarate, whereas the interaction was not influenced by the phosphorylation status of PII. Northern blot analyses revealed that the transcripts of a set of nitrogen-related genes, including nblA, nrtABCD, and ureG, were decreased in a pamA deletion mutant. The mRNA and protein levels of a group 2 sigma factor SigE were also reduced by the pamA mutation, and transcripts for sugar catabolic genes, such as gap1, zwf, and gnd that are under the control of SigE, were consequently decreased in the pamA mutant. In addition, the pamA mutant was found to be unable to grow in glucose-containing media. These results indicate that PamA has a role in the transcript control of genes for nitrogen and sugar metabolism in Synechocystis sp. PCC 6803.

Crystal Structure and Functional Analysis of Lipoamide Dehydrogenase from Mycobacterium tuberculosis

We report the 2.4 A crystal structure for lipoamide dehydrogenase encoded by lpdC from Mycobacterium tuberculosis. Based on the Lpd structure and sequence alignment between bacterial and eukaryotic Lpd sequences, we generated single point mutations in Lpd and assayed the resulting proteins for their ability to catalyze lipoamide reduction/oxidation alone and in complex with other proteins that participate in pyruvate dehydrogenase and peroxidase activities. The results suggest that amino acid residues conserved in mycobacterial species but not conserved in eukaryotic Lpd family members modulate either or both activities and include Arg-93, His-98, Lys-103, and His-386. In addition, Arg-93 and His-386 are involved in forming both "open" and "closed" active site conformations, suggesting that these residues play a role in dynamically regulating Lpd function. Taken together, these data suggest protein surfaces that should be considered while developing strategies for inhibiting this enzyme.

N1-Aminopropylagmatine, a New Polyamine Produced as a Key Intermediate in Polyamine Biosynthesis of an Extreme Thermophile, Thermus thermophilus

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.

Expression and refolding of tobacco anionic peroxidase from E. coli inclusion bodies.

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.