Effect of periplasmic expression of recombinant mouse interleukin-4 on hydrogen peroxide concentration and catalase activity in Escherichia coli

Oxidative stress occurs as a result of imbalance between generation and detoxification of reactive oxygen species (ROS). This kind of stress was rarely discussed in connection with foreign protein production in Escherichia coli. Relation between cytoplasmic recombinant protein expression with H₂O₂ concentration and catalase activity variation was already reported. The periplasmic space of E. coli has different oxidative environment in relative to cytoplasm and there are some benefits in periplasmic expression of recombinant proteins. In this study, hydrogen peroxide concentration and catalase activity following periplasmic expression of mouse IL-4 were measured in E. coli. After construction of pET2mIL4 plasmid, the expression of recombinant mouse interleukin-4 (mIL-4) was confirmed. Then, the H₂O₂ concentration and catalase activity variation in the cells were studied in exponential and stationary phases at various ODs and were compared to those of wild type cells and empty vector transformed cells. It was revealed that empty vector introduction and periplasmic recombinant protein expression increased significantly the H₂O₂ concentration of the cells. However, the H₂O₂ concentration in mIL-4 expressing cells was significantly higher than its concentration in empty vector transformed cells, demonstrating more effects of recombinant mIL-4 expression on H₂O₂ elevation. Likewise, although catalase activity was reduced in foreign DNA introduced cells, it was more lowered following expression of recombinant proteins. Correlation between H₂O₂ concentration elevation and catalase activity reduction with cell growth depletion is also demonstrated. It was also found that recombinant protein expression results in cell size increase.     

The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.)

To access contributions of inductive responses of the antioxidant enzymes in the resistance to salt stress, activities of the enzymes were determined in the rice (Oryza sativaL. cv. Dongjin) plant. In the leaves of the rice plant, salt stress preferentially enhanced the content of H₂O₂ as well as the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. On the other hand, salt stress had little effect on the activity levels of glutathione reductase (GR). In order to analyze the changes of antioxidant enzyme isoforms against salt stress, plant extracts were subjected to native PAGE. Leaves of the rice plant had two isoforms of Mn-SOD and five isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in the activity gels. Expression of Cu/Zn-1, -2, and Mn-SOD-2 isoforms was preferentially enhanced by salt stress. Seven APX isoforms were presented in the leaves of the rice plants. The intensities of APX-4 to -7 were enhanced by salt stress, whereas those of APX-1 to -3 were minimally in changed response to salt stress. There were seven GR isoforms in the leaves of rice plants. Levels of activity for most GR isoforms did not change in the stressed plants compared to the control plants. On the other hand, the levels of activity for most antioxidant enzymes changed little in the roots of stressed plants compared to the control plants. These results collectively suggest that SOD leads to the overproduction of hydrogen peroxide in the leaves of rice plants subjected to salt stress: The overproduction of hydrogen peroxide functions as the signal of salt stress, which induces the induction of specific APX isoforms but not specific GR isoforms under catalase deactivation.     

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H₂O₂) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (P_n) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.     

Overexpression of GASA5 increases the sensitivity of Arabidopsis to heat stress

Basal thermotolerance is very important for plant growth and development when plants are subjected to heat stress. However, little is known about the functional mechanism of gibberellins (GAs) in the basal thermotolerance of plants. In the present work, we provide molecular evidence that a member of the gene family encoding the GA-stimulated Arabidopsis (GASA) peptides, namely GASA5, is involved in the regulation of seedling thermotolerance. The GASA5-overexpressing plants displayed a weak thermotolerance, with a faster cotyledon-yellowing rate, lower seedling-survival rate, and slower hypocotyl elongation, in comparison to the wild-type and GASA5 null-mutant (gasa5-1) plants, after heat-stress treatment. The short-hypocotyl phenotype of GASA5-overexpressing plants could be rescued by the exogenous application of salicylic acid (SA), the hormone found to protect plants from heat stress-induced damage. GASA5 expression was inhibited by heat stress but unaffected by the application of exogenous SA. However, expression of the gene encoding the noexpresser of PR genes 1 (NPR1), a key component of the SA-signaling pathway, was downregulated by GASA5 overexpression. Importantly, when different GASA5-genotype plants were treated with heat stress, several genes encoding heat-shock proteins, including HSP101, HSP70B, HSP90.1, HSP17.6-C1, and HSP60, were inhibited by GASA5 overexpression. Meanwhile, hydrogen peroxide was accumulated at high levels in heat stress-treated GASA5-overexpressing plants. These results suggest that the Arabidopsis GASA5 gene acts as a negative regulator in thermotolerance by regulating both SA signaling and heat shock-protein accumulation.     

Enhancement of salt tolerance in transgenic rice expressing an Escherichia coli catalase gene, katE

Rice (Oryza sativa) is sensitive to salt stresses and cannot survive under low salt conditions, such as 50 mM NaCl. In an attempt to improve salt tolerance of rice, we introduced katE, a catalase gene of Escherichia coli, into japonica rice cultivar, Nipponbare. The resultant transgenic rice plants constitutively expressing katE were able to grow for more than 14 days in the presence of 250 mM NaCl, and were able to form flower and produce seeds in the presence of 100 mM NaCl. Catalase activity in the transgenic rice plants was 1.5- to 2.5-fold higher than non-transgenic rice plants. Our results clearly indicate that simple genetic modification of rice to express E. coli-derived catalase can efficiently increase its tolerance against salt stresses. The transformant presented here is one of the most salt-tolerant rice plants created by molecular breeding so far.     

Expression and purification of recombinant hemoglobin I from Lucina pectinata

Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric sulfide complex needed to transport H₂S to the bacterial endosymbiont. To further study HbI, expression studies of this protein were performed in Escherichia coli. This is the first time that the recombinant HbI was produced using a recombinant DNA expression system. Hemoglobin I cDNA was amplified and cloned into the TOPO-P_BAD expression vector, which contains a fusion tag of six histidine residues (6XHis tag). Plasmid clone sequence analysis was carried out in order to ensure that the insert was in the correct reading frame for proper protein expression in E. coli. The expression of recombinant HbI was optimal when induced for 5 hr with 0.002% of l-arabinose as detected by Western blot analysis. The proto-porphyrin group was inserted into the recombinant HbI. Purification of the heme-bound recombinant protein was performed under native conditions by affinity chromatography using Ni-NTA and Probond resins. The sodium dithionite-reduced recombinant protein presented a shift from the Soret band at 413-435 nm, indicating the presence of the heme group in the adequate amino acid environment of HbI. These results indicate that recombinant HbI from Lucina pectinata can be successfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding.     

Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (?uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ?uspA E. coli strain. The IPTG induced expression of a 56.6 kDa GST fusion protein was visualized on SDS–PAGE and attested by immunoblotting. E. coli ?uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl₂, H₂O₂, UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl₂ demonstrated about 22.6–51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.     

Metabolic Engineering for Resveratrol Derivative Biosynthesis in Escherichia coli

We previously reported that the SbROMT3syn recombinant protein catalyzes the production of the methylated resveratrol derivatives pinostilbene and pterostilbene by methylating substrate resveratrol in recombinant E. coli. To further study the production of stilbene compounds in E. coli by the expression of enzymes involved in stilbene biosynthesis, we isolated three stilbene synthase (STS) genes from rhubarb, peanut, and grape as well as two resveratrol O-methyltransferase (ROMT) genes from grape and sorghum. The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes. Out of three STS, only AhSTS was able to produce resveratrol from p-coumaric acid. Thus, to improve the solubility of RpSTS, VrROMT, and SbROMT3 in E. coli, we synthesized the RpSTS, VrROMT and SbROMT3 genes following codon-optimization and expressed one or both genes together with the cinnamate/4-coumarate:coenzyme A ligase (CCL) gene from Streptomyces coelicolor. Our HPLC and LC-MS analyses showed that recombinant E. coli expressing both ScCCL and RpSTSsyn led to the production of resveratrol when p-coumaric acid was used as the precursor. In addition, incorporation of SbROMT3syn in recombinant E. coli cells produced resveratrol and its mono-methylated derivative, pinostilbene, as the major products from p-coumaric acid. However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes. These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.     

Protein-Precursor tRNA Contact Leads to Sequence-Specific Recognition of 5′ Leaders by Bacterial Ribonuclease P

Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5′ leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5′ side of the cleavage site (N(− 4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(− 4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(− 4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(− 4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(− 4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(− 4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(− 4) that enhances RNase P affinity. This observation suggests that specificity at N(− 4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.     

E. coli sabotages the in vivo production of O-linked β-N-acetylglucosamine-modified proteins

The O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli β-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering β-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-β1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-β-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.     

The dihydrolipoyl acyltransferase gene BCE2 participates in basal resistance against Phytophthora infestans in potato and Nicotiana benthamiana

Dihydrolipoyl acyltransferase (EC 2.3.1.12), a branched-chain α-ketoacid dehydrogenase E2 subunit (BCE2), catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. However, the role of BCE2 responding to biotic stress in plant is not clear. In this study, we cloned and characterized a BCE2 gene from potato, namely StBCE2, which was previously suggested to be involved in Phytophthora infestans–potato interaction. We found that the expression of StBCE2 was strongly induced by both P. infestans isolate HB09-14-2 and salicylic acid. Besides, when the homolog of StBCE2 in Nicotiana benthamiana named NbBCE2 was silenced, plants showed increased susceptibility to P. infestans and reduced accumulation of hydrogen peroxide (H₂O₂). Furthermore, we found that a marker gene NbrbohB involved in the production of reactive oxygen species, was also suppressed in NbBCE2-silenced plants. However, silencing of NbBCE2 had no significant effect on the hypersensitive responses trigged by INF1, R3a-AVR3a^KI pair or Rpi-vnt1.1-AVR-vnt1.1 pair. Our results suggest that BCE2 is associated with the basal resistance to P. infestans by regulating H₂O₂ production.     

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

The response to oxidative stress induced by magnesium deficiency in kidney bean plants

To understand the plant response to oxidative stresses, we studied the influence of magnesium (Mg⁺⁺) deficiency on the formation of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and protease activity in kidney bean plants. The expression pattern of proteins under Mg⁺⁺ deficiency also was examined via two-dimensional electrophoresis. The formation of H₂O₂ and MDA increased in the primary leaves of plants grown in a nutrient solution deficient in Mg⁺⁺. Protease activity in Mg⁺⁺-deficient plants was also higher than in those grown with sufficient Mg⁺⁺. The expression pattern of the proteins showed that 25 new proteins were generated and 64 proteins disappeared under Mg⁺⁺-deficient conditions. Therefore, a deficiency in Mg⁺⁺ may cause oxidative stress and a change in protein expression. Some of these proteins may be related to the oxidative stress induced by Mg⁺⁺ deficiency.     

Salicylic acid and salicylic acid sensitive and insensitive catalases in different genotypes of chickpea against Fusarium oxysporum f. sp. ciceri

Differential expression of catalase isozymes in different genotypes of chickpea resistant genotypes- A1, JG-315, JG-11, WR-315, R₁-315, Vijaya, ICCV-15017, GBS-964, GBM-10, and susceptible genotypes- JG-62, MNK, ICCV-08321, ICCV-08311, KW-104, ICCV-08123, ICC-4951, ICC-11322, ICC-08116 for wilt disease caused by Fusarium oxysporum. f. sp. ciceri (Foc) was analyzed. Salicylic acid (SA) and H₂O₂ concentrations were determined in control as well as in plants infected with F. ciceri and found that the high and low levels of salicylic acid and H₂O₂ in resistant and susceptible genotypes of chickpea respectively. Catalase isozyme activities were detected in the gel and found that no induction of new catalases was observed in all the resistant genotypes and their some of the native catalase isozymes were inhibited; whereas, induction of multiple catalase isozymes was observed in all the screened susceptible genotypes and their activities were not inhibited upon Foc or SA treatments. The above results support the possible role of these isozymes as a marker to identify which genotype of chickpea is expressing systemic acquired resistance.