Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B

Insulin is a polypeptide hormone which is produced by the β‐cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des‐threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des‐threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2–3 at 25 °C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des‐threonine insulin and hence increased the production yield of active insulin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli.

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.     

Direct effect of glucose on the preproinsulin mRNA level in isolated pancreatic islets

The effects of glucose on the preproinsulin mRNA level and the rate of (pro)insulin biosynthesis were examined in isolated mouse pancreatic islets. Relative concentrations of preproinsulin mRNA were quantitated by a RNA-dot hybridization procedure. The level of preproinsulin mRNA in islets incubated for up to 7 days at 20 mM glucose remained constant. In islets incubated at 3.3 mM glucose the preproinsulin mRNA level decreased and was after 24 h reduced to one tenth of the level at 20 mM glucose. Subsequent incubation at 20 mM glucose completely restored the preproinsulin mRNA level but only after 3 days of culture, while the insulin release was restored within 24 h. The insulin-biosynthetic activity of the islets was correlated to the variation in the level of the preproinsulin mRNA. These results suggest that glucose does have a direct influence on the level of preproinsulin mRNA and that the rate of (pro)insulin biosynthesis is limited by the level of the preproinsulin mRNA.     

Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli

Lipase (EC 3.1.1.3) is a popular enzyme used as an ingredient in detergents and biocatalyst in many biochemical reactions. Lipase is usually expressed in Escherichia coli as an inactive inclusion body and at a low level. In this study, Candida antarctica lipase B (CalB) was fused with various polycationic amino acid tags and expressed in E. coli in order to increase a soluble expression level. By induction with 1.0 mM IPTG, the authentic and fused CalBs were expressed at 27-56% of total protein. The 10-arginine and 10-lysine tags fused at the C-terminal of CalB significantly increased the solubility of CalB by five- to ninefold, relative to the case of the authentic CalB expressed in a recombinant E. coli Origami 2? (DE3) strain. Among a series of the C-terminal poly-arginine tags, the recombinant CalB combined with the 10-arginine tag (CalB-R10) possessed the highest lipase specific activity of 9.5 ± 0.03 U/mg protein, corresponding to a fourfold enhancement compared with the authentic CalB.     

Production and characterization of bioactive recombinant resistin in Escherichia coli

Type 2 diabetes, characterized by peripheral target tissue resistance to insulin, is epidemic in industrialized countries and is strongly associated with obesity. The protein hormone, resistin, secreted specifically by the adipose tissues, is found to antagonize insulin action upon glucose uptake and may serve as an important role between human obesity and insulin resistance. Here, we report the production of bioactive recombinant resistin in Escherichia coli. cDNA of resistin was obtained by RT-PCR from mRNA of mouse differentiated NIH/3T3-L1 cells. The cDNA of mature resistin was inserted in the pQE-31 vector and the recombinant plasmid was transferred into E. coli JM109. After IPTG induction, the rec. resistin found in the inclusion body was dissolved in 6 M guanidine-HCl in the presence of 10 mM beta-mercaptoethanol. The His-tag containing protein was purified by Ni-NTA column to 95% homogeneity. After a quasi-static-like refolding process, the secondary structure of the rec. resistin was elucidated by circular dichroism which indicated that the protein was composed of 34.3% alpha-helix, 8.9% beta-sheet, 23.4% beta-turn, and 31.2% unordered structure. No disulfide-linked homodimers were formed in SDS-PAGE analysis under non-reducing conditions. The rec. resistin showed a dose-dependent antagonizing action against insulin in [3H]-2-deoxy-glucose transport in a broad range from 1 ng ml(-1) to 10 microg ml(-1) of resistin. A suppression of 85% of transport was achieved at the dosage of 10 microg ml(-1). This result may indicate that the rec. resistin does not need to form homodimers to establish its bioactivity. The rec. resistin will be useful for exploring the biological functions of this newly discovered hormone.     

Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose.

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.     

hGH-双精氨酸C肽人胰岛素原基因的克隆表达及纯化研究

旨在提高基因重组人胰岛素在大肠杆菌中表达的稳定性及表达包涵体蛋白的复性水平.在人胰岛素原N端前融合人生长素N端的一段序列来充当前导肽,同时将C肽设计为两个精氨酸,分10段合成长链寡核苷酸链,利用重叠延伸PCR技术(SOE PCR)扩增得到该基因片段.与表达载体PET-30a连接,转化E.coli BL21(DE3),IPTG诱导表达.表达的融合蛋白采用Ni-NTA亲和层析纯化,纯化后的蛋白经复性、冻干等步骤后用胰蛋白酶,羧肽酶B双酶切再过DEAE Sepharose Fast Flow阴离子交换柱,收集洗脱峰.对制备所得的胰岛素用SDS-PAGE,Western blot进行性质鉴定,及皮下注射小鼠测定生物活性.结果显示,目的蛋白在大肠杆菌BL21(DE3)中得到了表达,表达产物以不溶性包涵体形式纯在,约占大肠杆菌总蛋白的30%.经Ni-NTA亲和层析得到的重组蛋白纯度为85%,DEAE Sepharose Fast Flow阴离子交换纯化得到单组分胰岛素.Western Blot显示制备所得的胰岛素具有胰岛素免疫原性,皮下给药注射小鼠活性测定表明具有明显的降血糖活性.获得了一种高效生产基因重组人胰岛素的方法,为研究胰岛素类似物奠定了前期基础同时也为今后探索胰岛素的非注射给药途径提供了原料.     

Met-Lys-双C肽人胰岛素原基因的构建表达及分离纯化

应用 Ｐ Ｃ Ｒ 定点突变方法构建编码 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原基因,并在大肠杆菌中以包含体方式获得表达 表达产物经还原、重组、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 分离纯化,获得 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原,经胰蛋白酶与羧肽酶 Ｂ的酶解, Ｒｅｓｏｕｒｃｅ Ｔ Ｍ Ｑ 阴离子交换柱层析分离制备得人胰岛素,其放免活性、受体结合活性均与猪胰岛素相同      

Synthesis of genistein derivatives and determination of their protective effects against vascular endothelial cell damages caused by hydrogen peroxide

A series of genistein derivatives, prepared by alkylation and difluoromethylation, were tested for their inhibitory effects on the hydrogen peroxide induced impairment in human umbilical vein endothelial (HUVE-12) cells in vitro. The HUVE-12 cells were pretreated with either the vehicle solvent (DMSO), genistein, or different amounts of the genistein derivatives for 30 min before exposed to 1 mM hydrogen peroxide for 24 h. Cell apoptosis was determined by flow cytometry with propidium iodide (PI) staining. Cellular injury was estimated by measuring the lactate dehydrogenase (LDH) release. Data suggested that the genistein derivatives possessed a protective effect on HUVE-12 cells from hydrogen peroxide induced apoptosis and reduced LDH release. Among these derivatives, 7-difluoromethyl-5,4'-dimethoxygenistein exhibited the strongest activity against hydrogen peroxide induced apoptosis of HUVE-12 cells.     

Role of hydrogen peroxide in loss of culturability mediated by visible light in Escherichia coli in a freshwater ecosystem.

A study was made of the mechanisms by which visible light produces cell dormancy in Escherichia coli, resulting in loss of culturability. Visible light may act directly on the cells or generate photoproducts with a negative effect on the cells. In nonilluminated microcosms the addition of increasing concentrations of hydrogen peroxide, one of the photoproducts formed in natural aquatic systems, gave rise to the formation of nonculturable cells and injured culturable cells, and this negative effect depended on the concentration of peroxide. On the other hand, in illuminated microcosms the addition of compounds which eliminate hydrogen peroxide (i.e., catalase, sodium pyruvate, and thioglycolate) had a protective effect on the E. coli cells, as the CFU counts on minimal medium and on recuperation medium were significantly higher (P < 0.05) than those detected in the absence of these compounds. Furthermore, when hydrogen peroxide was eliminated, the CFU counts on recuperation medium did not fall significantly, indicating that nonculturable cells did not form. These results rule out the direct effect of visible light on the cells and show that hydrogen peroxide, generated photochemically, may be the cause of the loss of culturability of E. coli in illuminated systems.     

Role of hydrogen peroxide in loss of culturability mediated by visible light in Escherichia coli in a freshwater ecosystem.

A study was made of the mechanisms by which visible light produces cell dormancy in Escherichia coli, resulting in loss of culturability. Visible light may act directly on the cells or generate photoproducts with a negative effect on the cells. In nonilluminated microcosms the addition of increasing concentrations of hydrogen peroxide, one of the photoproducts formed in natural aquatic systems, gave rise to the formation of nonculturable cells and injured culturable cells, and this negative effect depended on the concentration of peroxide. On the other hand, in illuminated microcosms the addition of compounds which eliminate hydrogen peroxide (i.e., catalase, sodium pyruvate, and thioglycolate) had a protective effect on the E. coli cells, as the CFU counts on minimal medium and on recuperation medium were significantly higher (P < 0.05) than those detected in the absence of these compounds. Furthermore, when hydrogen peroxide was eliminated, the CFU counts on recuperation medium did not fall significantly, indicating that nonculturable cells did not form. These results rule out the direct effect of visible light on the cells and show that hydrogen peroxide, generated photochemically, may be the cause of the loss of culturability of E. coli in illuminated systems.     

Reactive oxygen-reducing and protein-refolding activities of adult T cell leukemia-derived factor/human thioredoxin.

Reducing and protein-refolding activities of adult T cell leukemia-derived factor (ADF)/human thioredoxin were studied. Recombinant ADF/human thioredoxin produced by E. coli, which has an insulin-reducing activity as efficient as that of E. coli thioredoxin, also reduced some reactive oxygen species, such as hydrogen peroxide. Furthermore, recombinant ADF/human thioredoxin was found to have protein-refolding activity for scrambled (mispaired disulfide-containing) RNase A. Cys-31 at the active site of ADF/human thioredoxin proved essential for reducing activity, and loss of Cys-31 in ADF/human thioredoxin attenuated the protein-refolding activity. These data suggest a physiological role of ADF/human thioredoxin in protecting living cells from proteotoxicity caused by reactive oxygens in vivo.     

The effect of human serum transferrin and milk lactoferrin on hydroxyl radical formation from superoxide and hydrogen peroxide

The effect of transferrins on hydroxyl radical formation from the superoxide anion and hydrogen peroxide generated by the xanthine-xanthine oxidase system has been studied by EPR using 5,5-dimethyl-1-pyrroline N-oxide as a spin trap. Neither diferriclactoferrin nor diferrictransferrin were found capable of promoting hydroxyl radical formation via the Haber-Weiss reaction even in the presence of EDTA in concentrations up to 1 mM. Activity observed by other authors may have been due to the presence of extraneous iron or an active protein impurity. Partially saturated transferrin and lactoferrin present in normal subjects may protect cells from damage by binding iron that might catalyze hydroxyl radical formation from superoxide and hydrogen peroxide. In any event, the hydroxyl radical formation observed in active neutrophils during phagocytosis cannot be associated with lactoferrin activity.     

Characterization of acatalasemic erythrocytes treated with low and high dose hydrogen peroxide. Hemolysis and aggregation

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mM hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mM, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (*Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.     

Properties of 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate--an intermediate in the non-mevalonate isoprenoid biosynthesis

Extraction and purification from the biomass of Corynebacterium ammoniagenes of 2-C-methyl-D-erhythritol 2,4-cyclopyrophosphate (MEC) was associated with its spontaneous transformation into a number of derivatives (which was due to pyrophosphate bond lability and the formation of complexes with metals). These derivatives included 1,2-cyclophospho-4-phosphate, 2,4-diphosphate, 2,3-cyclophosphate, 1,4-diphosphate, and 3,5-diphosphate (identified by 1H, 31P, and 13C NMR spectroscopy) and accounted for about 10% MEC. When added to a solution of DNA in the presence of the Fenton reagent, MEC prevented DNA decomposition. In addition, MEC slowed down the interaction of the reagent with tempol radicals, which indicates that complexation of ferrous ions by MEC attenuates their ability to catalyze the formation of hydroxyl radicals from hydrogen peroxide. In the presence of 0.23 mM MEC, the rate of respiration of rat liver mitochondria increased 1.8 times. At 0.1-1.0 mM, MEC activated in vitro proliferation of human Vgamma9 T-cells. It is suggested that MEC acts as an endogenous stabilizing agent for bacterial cells subjected to oxidative stress and as an immunomodulator for eukaryotic hosts.     

Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.     

Runx2‐mediated bcl‐2 gene expression contributes to nitric oxide protection against hydrogen peroxide‐induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. This study was aimed to evaluate the protective effects of pretreatment with sodium nitroprusside (SNP) as a source of NO on hydrogen peroxide‐induced osteoblast insults and its possible mechanisms. Exposure of human osteosarcoma MG63 cells to hydrogen peroxide significantly increased cellular oxidative stress, but decreased ALP activity and cell viability, inducing cell apoptosis. Pretreatment with 0.3 mM SNP significantly lowered hydrogen peroxide‐induced cell insults. Treatment of human MG63 cells with hydrogen peroxide inhibited Bcl‐2 mRNA and protein production, but pretreatment with 0.3 mM SNP significantly ameliorated such inhibition. Sequentially, hydrogen peroxide decreased the mitochondrial membrane potential, but increased the levels of cytochrome c and caspase‐3 activity. Pretreatment with 0.3 mM SNP significantly lowered such alterations. Exposure to hydrogen peroxide decreased Runx2 mRNA and protein syntheses. However, pretreatment with 0.3 mM SNP significantly lowered the suppressive effects. Runx2 knockdown using RNA interference inhibited Bcl‐2 mRNA production in human MG63 cells. Protection of pretreatment with 0.3 mM SNP against hydrogen peroxide‐induced alterations in ALP activity, caspase‐3 activity, apoptotic cells, and cell viability were also alleviated after administration of Runx2 small interference RNA. Thus, this study shows that pretreatment with 0.3 mM SNP can protect human MG63 cells from hydrogen peroxide‐induced apoptotic insults possibly via Runx2‐involved regulation of bcl‐2 gene expression. J. Cell. Biochem. 108: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.     

Visible light damage to Escherichia coli in seawater: oxidative stress hypothesis

The effect of visible light on Escherichia coli H10407 in seawater microcosms was investigated. Light damage was estimated by loss of colony-forming ability. Illumination of E. coli suspended in oligotrophic seawater with visible light at an intensity of about 40 klux caused a drastic decrease of culturable bacteria which turned to a viable but non-culturable state. In seawater E. coli exhibited weak metabolic activity as estimated by ³H methyl-thymidine incorporation in the cell. Visible light did not significantly alter this metabolic activity and did not involve detectable oxidation of lipid membranes as evaluated by gas chromatography analysis of fatty acids. The involvement of oxygen and reactive oxygen species in phototoxicity was studied. A decrease of the toxic effect was observed when E. coli was exposed to visible light under anaerobic conditions. Scavengers of reactive oxygen species exhibited variable protective effects. β-Carotene, a singlet oxygen scavenger, and superoxide dismutase were equally ineffective. On the other hand, catalase, which eliminates hydrogen peroxide and thiourea, a hydroxyl radical scavenger, showed a net protection. In addition desferrioxamine B, an iron chelator, was also effective in reducing phototoxicity, probably by preventing hydroxyl radical generation by decomposition of hydrogen peroxide in the presence of iron (Fenton reaction). Therefore, hydrogen peroxide and hydroxyl radical seem to be reactive intermediates of oxygen-dependent (type II) photosensitized reactions.     

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.