Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.     

Effect of polymers on enhanced chemiluminescent assays for peroxidase and peroxidase labels

Hydroxypropyl methylcellulose, hydroxyethyl cellulose, and hydroxybutyl methylcellulose stabilized light emission in a boronic acid-enhanced chemiluminescent assay for horseradish peroxidase. The stabilization of light emission was concentration-dependent and more effective with substituted boronic acid enhancers (e.g. 4-iodophenylboronic acid) than with substituted phenol enhancers (e.g. 4-iodophenol). Hydroxybutyl methylcellulose improved the linearity of the dose–response curve in a peroxidase-based antioxidant assay and stabilized light emission post-consumption of the antioxidant (Trolox). This polymer had no effect on the signal from a peroxidase label immobilized on a membrane (dot blot) or on the inside surface of a microwell in an enzyme immunoassay for thyrotropin.     

New enhancers for the chemiluminescent peroxidase catalysed chemiluminescent oxidation of pyrogallol and purpurogallin

The effects of various boronate compounds, 4-biphenylboronic acid, 4-bromobenzeneboronic acid, trans-4-(3-propionic acid)phenylboronic acid and 4-iodophenylboronic acid, on the horseradish peroxidase (HRP) catalysed chemiluminescent oxidation of pyrogallol and purpurogallin by peroxide were investigated. trans-4-(3-Propionic acid)phenylboronic acid produced a 13.7-fold enhancement in the peak light emission from the chemiluminescent HRP catalysed pyrogallol reaction (detection limit for HRP < 1.25 fmol). At low enhancer concentration a single peak of light emission was observed and as the enhancer concentration increased the time to peak light emission became progressively longer. The chemiluminescence showed two peaks at higher concentrations (> 54.3 μmol/L) and the individual peak times depended upon the concentration of the enhancer. All of the boronates enhanced peak light emission in the chemiluminescent HRP catalysed purpurogallin reaction. 4-Biphenylboronic acid was the most effective and it enhanced peak light emission 314-fold. The practical detection limit for HRP (Type VIA) using this enhancer was 4.18 pmol (peak emission at 20 minutes). This compound also enhanced peak light emission 232-fold from a chemiluminescent HRP-purpurogallin reaction in which molecular oxygen replaced peroxide as the oxidant.     

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.     

Effects of mutations in the helix G region of horseradish peroxidase

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F' (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to 2-fold) and solvents (up to 4-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic. Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state V(m)/E values occurred with reducing substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), despite the distance of the mutated positions from the active site.     

4-phenylylboronic acid: A new type of enhancer for the horseradish peroxidase catalysed chemiluminescent oxidation of luminol

4-Phenylylboronic acid enhances the light emission from the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. Optimization studies showed that the greatest enhancement was obtained using micromolar concentrations of the new enhancer. The largest degree of enhancement was found with the basic isoenzyme of horseradish peroxidase (Type VIA), and lesser degrees of enhancement were obtained with Type VII and Type IX horseradish peroxidase. The enhancer was also effective in the peroxidase catalysed oxidation of isoluminol by peroxide.     

Decreased peroxidase activity in transgenic tobacco and its effect on lignification

An antisense gene construct of a peroxidase gene (Shpx6a) from a tropical pasture legume Stylosanthes humilis was transferred into tobacco cells via Agrobacterium tumefaciens to test whether peroxidase activity could be decreased and what effect this would have on lignification. A large number of tobacco cell lines were regenerated on selective media and stable integration of the transgene was confirmed in randomly selected putative transformants. Analyses of the primary transgenic plants and their progeny (T ₁) demonstrated that the total peroxidase activity was significantly decreased (up to 36%) as compared to that measured in untransformed control plants. Importantly, reduction in peroxidase activity is accompanied by decreases (up to 23%) in lignin content in several transgenic lines.     

Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T₀ and T₁ populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.     

Hydroxyproline-O-glycosylated peptide tags enhance recombinant protein yields in tobacco transient expression

Plant transient expression provides a rapid production platform for recombinant proteins but is linked with low protein yields. To test if plant-specific hydroxyproline (Hyp)-O-glycosylated peptide tags attached to a target protein can improve overall yields of recombinant protein transiently expressed in Nicotiana benthamiana, enhanced green fluorescence protein (EGFP) was expressed as a fusion with 5 or 32 tandem repeats of a serine–proline motif, designated (SP)₅ or (SP)₃₂, which is known to direct extensive Hyp-O-glycosylation in plants. EGFP containing the (SP)_n motif showed enhanced yields in the order as follows: EGFP < EGFP-(SP)₅ ≪ (SP)₅-EGFP < (SP)₃₂-EGFP. The EGFP equivalent yield of (SP)₃₂-EGFP was up to 16-fold greater than that of the EGFP control. In addition, both fully glycosylated (SP)₃₂-EGFP (∼115 kDa) and partially glycosylated (SP)₃₂-EGFP (∼40 kDa) were detected in protein extracts of N. benthamiana. These two types of glycoforms were completely segregated between media and cells in tobacco BY-2 cell cultures.     

Strategies for the suppression of peroxidase gene expression in tobacco. I. Designing efficient ribozymes

Five short hammerhead ribozymes (Rzs) were constructed and tested, using a range ofin vitro reaction conditions, for catalytic activity against the mRNA encoding the lignin-forming peroxidase (TPX) of tobacco. Although all 5 Rzs were shown to be able to cleave the RNA substrate, percentage cleavage varied with pre-denaturation of Rz and substrate, incubation temperature, length of incubation and ribozyme (Rz)-to-substrate ratio. One Rz with two catalytic units and 60 nucleotides of complementary sequence in 3 regions was shown to most efficiently cleave the substrate under allin vitro conditions tested. This ribozyme cleaved better than the two single ribozymes from which it was made. The superior cleaving ability of this Rz was shown to be due to the accessibility of the chosen target site and to the increased length of the hybridizing arms spanning this accessible region of the RNA.     

Cloning and expression of two genes encoding auxin-binding proteins from tobacco

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.     

具有抗HIV活性的突变型天花粉蛋白TCSYFF-KR的构建及原核表达

目的：构建具有抗HIV活性的突变型天花粉蛋白（TCS）,并将其在原核系统内进行表达与纯化。方法：借助计算机预测TCS分子上可能的抗原决定簇（YFF81-83和KR173-174）,并依此设计适当的突变引物;以栝楼基因组DNA为模板,利用重组PCR技术扩增双突变型TCS全长基因,经BamHI和EcoRI双酶切后与原核表达载体pRSET-A连接,转化感受态大肠杆菌DH5α,提取质粒进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21（DE3）,经IPTG诱导表达后,对表达产物进行Western印迹鉴定;用Ni-NTA亲和层析柱对所获突变型TCS蛋白进行纯化。结果：构建了突变型TCSYFY-KR,并获得了该蛋白在大肠杆菌内的可溶性高效表达;经Ni-NTA亲和层析柱纯化,产生大量均一的突变型TCS蛋白。结论：TCS的定点突变及其在原核系统内的表达,为基因工程方法改造TCS提供了一条新途径。     

Improvement of mimetic peroxidase activity of gold nanoclusters on the luminol chemiluminescence reaction by surface modification with ethanediamine

Peroxidase is a commonly used catalyst in luminol–H₂O₂ chemiluminescence (CL) reactions. Natural peroxidase has a sophisticated separation process, short shelf life and unstable activity, therefore it is important to develop peroxidases that have both high catalytic activity and good stability as alternatives to the natural enzyme. Gold nanoclusters (Au NCs) are an alternative peroxidase with catalytic activity in the luminol–H₂O₂ CL reaction. In the present study, ethanediamine was modified on the surface of Au NCs forming cationic Au NCs. The zeta potential of the cationic Au NCs maintained its positive charge when the pH of the solution was between 4 and 9. The cationic Au NCs showed higher catalytic activity in the luminol–H₂O₂ CL reaction than did unmodified Au NCs. A mechanism study showed that the better performance of cationic Au NCs may be attributed to the generation of ¹O₂ on the surface of cationic Au NCs and a positive surface charge, for better affinity to luminol. Cationic Au NC, acting as a peroxidase mimic, has much better stability than horseradish peroxidase over a wide range of temperatures. We believe that cationic Au NCs may be useful as an artificial peroxidase for a wide range of potential applications in CL and bioanalysis.     

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5-Srglb3-uidA-3nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.     

Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways

Versatile peroxidases (VP), a recently described family of ligninolytic peroxidases, show a hybrid molecular architecture combining different oxidation sites connected to the heme cofactor. High-resolution crystal structures as well as homology models of VP isoenzymes from the fungus Pleurotus eryngii revealed three possibilities for long-range electron transfer for the oxidation of high redox potential aromatic compounds. The possible pathways would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 of isoenzyme VPS1. These residues are exposed, and less than 11 A apart from the heme. With the purpose of investigating their functionality, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways in this isoenzyme; and (ii) incorporated the absent putative pathway. Analysis of the variants showed that Trp164 is required for oxidation of two high redox potential model substrates (veratryl alcohol and Reactive Black 5), whereas the two other pathways (starting at His232 and His82) are not involved in long-range electron transfer (LRET). None of the mutations affected Mn2+ oxidation, which would take place at the opposite side of the enzyme. Substitution of Trp164 by His also resulted in an inactive variant, indicating that an indole side-chain is required for activity. It is proposed that substrate oxidation occurs via a protein-based radical. For the first time in a ligninolytic peroxidase such an intermediate species could be detected by low-temperature electron paramagnetic resonance of H2O2-activated VP, and was found to exist at Trp164 as a neutral radical. The H2O2-activated VP was self-reduced in the absence of reducing substrates. Trp164 is also involved in this reaction, which in the W164S variant was blocked at the level of compound II. When analyzing VP crystal structures close to atomic resolution, no hydroxylation of the Trp164 Cbeta atom was observed (even after addition of several equivalents of H2O2). This is in contrast to lignin peroxidase Trp171. Analysis of the crystal structures of both peroxidases showed differences in the environment of the protein radical-forming residue that could affect its reactivity. These variations would also explain differences found for the oxidation of some high redox potential aromatic substrates.     

抗体重链可变区框架Ⅰ区对抗体分泌的影响

抗体重链可变区框架Ⅰ区(FR-Ⅰ)对抗体在原核细胞中的分泌表达具有显著的影响,单个氨基酸的改变即可导致抗体分子分泌能力的丧失.为了探索抗体在哺乳动物细胞中的高效表达,我们对一株不能有效分泌的人源抗狂犬病病毒抗体pCMV-RV/VH的FR-Ⅰ区氨基酸编码基因进行定点突变研究.实验显示,抗体重链可变区FR-Ⅰ区H6位的氨基酸由Glu突变为Gin之后,抗体的分泌表达水平得到了显著的提高,并且与抗原特异性结合的能力也明显增强.通过免疫荧光法对抗体在细胞内的转运过程进行了初步的探讨,发现能够有效分泌的抗体与无分泌表达的抗体都能够在细胞内进行正常的转录和翻译,进入内质网并转运至高尔基体,而且胞内表达水平基本一致.我们认为分泌能力的不同可能是FR-Ⅰ区影响抗体分子的折叠与装配所致,其中该区H6位氨基酸的性质能够显著影响抗体在哺乳动物细胞中的分泌.本研究以抗体重链可变区FR-Ⅰ区氨基酸为焦点,对基因工程抗体在真核细胞中高效表达的影响因素进行了探索,为改进抗体规模化生产提供了依据.     

Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

We characterized the biochemical functions of the small nonessential (C101-C104) and the large essential (C173-C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca(2+) protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca(2+), all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca(2+)-containing buffer. However, when diluted into a Ca(2+)-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65 degrees C, brDNase(C101A) at 60 degrees C, and brDNase(F192C/A217C) at 73 degrees C, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca(2+)-containing buffer, 10%-18% of the bpDNase activity was restored, suggesting that the "essential" disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the "nonessential" disulfide of bpDNase and the active-site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation (DeltaA650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction.     

有机磷降解酶在重组大肠杆菌中的表达及一步纯化固定化

【背景】有机磷化合物作为一类广谱杀虫剂,因其用量大、毒性强且不易降解,在自然界中的残留已对环境造成了严重污染。【目的】有机磷降解酶(organophosphohydrolase,OpdA)可降解多种有机磷化合物,探究其被固定到NiCo_2O_4载体上后用于有机磷化合物的降解效果。【方法】构建含组氨酸标签(histidine tag,His-tag)的OpdA,以pET-28a(+)为载体,Escherichia coli Rosetta(DE3)为宿主细胞,在终浓度为1.0 mmol/L的IPTG诱导下表达His-tagged OpdA。采用一步纯化固定化方法,实现固定化酶(OpdA@NiCo_2O_4)的制备。【结果】采用水热处理和煅烧制备了含过渡金属离子的NiCo_2O_4,利用过渡金属离子对酶分子表面组氨酸咪唑基的配位作用,实现了发酵粗酶液中His-taggedOpdA的一步纯化固定化,在优化条件下获得了高稳定性的OpdA@NiCo_2O_4;然后将其用于有机磷化合物的降解,在NaBH_4存在条件下,通过级联反应和降解条件优化,实现了有机磷化合物的高效降解。【结论】该研究不但实现了重组酶的一步分离纯化和固定化,也为有机磷化合物的降解提供了一条安全、高效、环保的新途径。