Production and Characterization of Hirudin Variant-1 by SUMO Fusion Technology in E. coli

Hirudin is the most potent non-covalent inhibitor of thrombin. Several expression systems have been used to produce recombinant hirudin for pharmaceutical purposes. However, high expression of active hirudin in Escherichia coli cytoplasm has not been successful owing to the fact that heterogenetic small peptide is easily degraded in the cell. To solve this problem, we constructed a recombinant form of the hirudin variant-1 (HV1) as a fusion protein with the small ubiquitin-related modifier gene (SUMO) by use of over-lap PCR. The fusion gene His₆-SUMO-HV1 was highly expressed in E. coli BL21 (DE3) in which the SUMO-HV1 accounts for over 30% of the soluble fraction. The fusion protein was purified by Ni?CNTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to release the HV1 with natural N-terminal. The recombinant HV1 (rHV1) was further purified by Ni?CNTA affinity chromatography and then by Q anion-exchange chromatography. N-terminal sequencing result demonstrated the purified rHV1 had the same N-terminal sequence as the native hirudin. MALDI-TOF/MS analysis indicated that the molecular weight of the purified rHV1 protein was 6939.161 Da, which was similar to the theoretical molecular weight of rHV1 6,944 Da. The Chromozym TH assay result showed that the anti-thrombin activity of purified rHV1 was 8,800 ATU/mg and comparable to the specific activity of native hirudin.     

重组水蛭素的纯化和鉴定

本文报导了化学合成的水蛭素基因在酵母细胞中得到表达,井能分泌水蛭素到胞外。将该菌株培养物的上清液经硫酸铵沉淀和Sephadex G-50过滤后,用DEAE-SephadexA-25进行阴离子交换层析,进而用HPLC反相层析,得到表达产物重组水蛭素。经SDS-PAGE,氨基酸序列分析,抗凝血酶活力分析及血浆滴定实验等方法鉴定,证明该基因表达产物与天然水蛭素HV_2相同。     

Biochemical and conformational characterization of a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2

In order to search for valuable and extremely thermo-stable enzymes that could be used in the protein hydrolysis industry, the gene corresponding to a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2 (GtLAP) was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined. Meanwhile, potential applications of GtLAP in the hydrolysis of anchovy proteins were also investigated. GtLAP was overexpressed in IPTG-induced E. coli BL21 (pET28a-LAP) as a soluble protein, and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 125?±?8.75 U/mg proteins. The molecular mass of GtLAP was estimated to be 55?kDa by SDS-PAGE analysis. The optimal reaction temperature and pH of GtLAP were 70?°C and 8.0, respectively. Under optimal conditions, GtLAP showed a marked preference for Leu-p-nitroanilide, followed by Met- and Phe-derivatives. Activity of GtLAP was strongly stimulated by Ni²⁺ ions, but was strongly inhibited by Hg²⁺. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of GtLAP to various extents and further induce changes in enzymatic activity. Results of hydrolytic experiment showed that combining GtLAP with endogenous enzymes could significantly increase the degree of hydrolysis to anchovy proteins and concentrations of free amino acids in hydrolysates. In this regard, GtLAP could potentially be used in the protein hydrolysis industry.     

Biochemical characterisation of two forms of halo- and thermo-tolerant chitinase C ofSalinivibrio costicola expressed inEscherichia coli

Two forms of chitinase C (Chi-I and Chi-II) were purified until homogeneity from the culture supernatant of a transformantEscherichia coli harbouringchitinase C gene from the halophilic bacteriumSalinivibrio costicola strain 5SM-1. Chi-II was derived from Chi-I by C-terminal processing. Chi-I and Chi-II showed similar salinity optimum at 1–2% NaCl and retained more than 80% of their activity at 3–5% NaCl and more than 50% residual activity at 14% NaCl. The two enzymes could also well function (activity > 95%) in the absence of NaCl. Both had highest activity at pH 7.0 and 50 °C and both were stable over a wide range of pH (3.0–10.0). More than 50% activity remained at 80 °C after 60 min treatment. Among 4 major cations contained in sea water, only Mg²⁺ at 10 mM increased activity about 10%. Usingp-nitrophenyl-N,N′-diacetylchitobiose as substrate, Chi-I and Chi-II hadK _m of 30 and 31.8 μM andV _max of 10 and 9.2 μmol/h/mg protein, respectively. Chi-I and Chi-II were classified as exochitinases by product analysis of theE. coli culture supernatant with high performance liquid chromatography (HPLC) and thin-layer chromatography (TLC).     

Comparative biochemical characterization and in silico analysis of novel lipases Lip11 and Lip12 with Lip2 from Yarrowia lipolytica

Novel lipases lip11 and lip12 from Yarrowia lipolytica MSR80 were cloned and expressed in E. coli HB101 pEZZ18 system along with lip2. These enzymes were constitutively expressed as extracellular proteins with IgG tag. The enzymes were purified by affinity chromatography and analyzed by SDS-PAGE with specific activity of 314, 352 and 198?U/mg for Lip2, Lip11 and Lip12, respectively on olive oil. Biochemical characterization showed that all were active over broad range of pH 4.0?C9.0 and temperature 20?C80?°C with optima at pH 7 and 40?°C. All the three lipases were thermostable up to 80?°C with varying t_1/2. Activity on various substrates revealed that they were most active on oils?>?triacylglycerides?>?p-np-esters. Relatively Lip2 and Lip11 showed specificity for mid to long chain fatty acids, while Lip12 was mid chain specific. GC analysis of triolein hydrolysis by these lipases revealed that Lip2 and Lip11 are regioselective, while Lip12 is not. Effect of metal ions showed that Lip2 and Lip12 were activated by Ca²⁺ whereas Lip11 by Mg²⁺. All were thiol activated and inhibited by PMSF and N-bromosuccinimide. All were activated by non polar solvents and inhibited by polar solvents. Detailed sequence analysis and structural predictions revealed Lip11 and Lip12 shared 61 and 62?% homology with Lip2 (3O0D) and three dimensional superimposition revealed Lip2 was closer to Lip11 than to Lip12 as was observed during biochemical characterization. Finally, thermostability and substrate specificity has been explained on the basis of detailed amino acid analysis.     

Bacterial inoculation speeds zinc release from ground tire rubber used as Zn fertilizer for corn and sunflower in a calcareous soil

Background and Aims

We tested the utility of some biological treatments to hasten degradation of waste tire rubber in soil and thus the release of zinc and sulfur for plant uptake.

Methods

Three rates of ground tire rubber (0, 150, and 300?mg?kg^?1) were incorporated into a Zn-deficient calcareous soil. Before addition to the soil, ground rubber was given four microbial treatments including no inoculation, inoculation with Rhodococcus erythropolis, inoculation with R. erythropolis+Escherichia coli, and inoculation with R. erythropolis+E. coli+Acinobacter calcoaceticus. In the pot experiment, corn (Zea mays L. Hybrid Single Cross 500) and sunflower (Helianthus annuus L. cv. Record) plants were exposed to three rates of ground rubber (0, 150, and 300?mg?kg^?1) or 3?mg zinc kg^?1 as ZnSO₄. Before addition to the soil, ground rubber and ZnSO₄ were inoculated or non-inoculated with R. erythropolis+E. coli+A. calcoaceticus.

Results

Ground rubber and microbial inoculation treatments reduced soil pH and the magnitude of this reduction increased over time. Ground rubber in combination with microbial inoculation increased DTPA-extractable soil Zn and Fe. The amount of DTPA-extractable Zn and Fe of rubber-amended soils increased over time so that the highest concentration of available Zn and Fe was found at week 10. Application of microbial inoculated ground tire rubber significantly increased shoot Zn concentration of each plant species.

Conclusions

Bacterial inoculation of ground rubber was effective in hastening increase in DTPA-extractable Zn in the studied calcareous soil and in enhancing Zn uptake by plants.     

Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization

Enhancing the production of α-cyclodextrin glycosyltransferase (α-CGTase) is a key aim in α-CGTase industries. Here, the mature α-cgt gene from Paenibacillus macerans JFB05-01 was redesigned with systematic codon optimization to preferentially match codon frequencies of Escherichia coli without altering the amino acid sequence. Following synthesis, codon-optimized α-cgt (coα-cgt) and wild-type α-cgt (wtα-cgt) genes were cloned into pET-20b(+) and expressed in E.?coli BL21(DE3). The total protein yield of the synthetic gene was greater than wtα-cgt expression (1,710?mg?L^?1) by 2,520?mg?L^?1, with the extracellular enzyme activity being improved to 55.3?U?mL^?1 in flask fermentation. ΔG values at -3 to +50 of the pelB site of both genes were ?19.10?kcal?mol^?1. Functionally, coα-CGTase was equally as effective as wtα-CGTase in forming α-cyclodextrin (α-CD). These findings suggest that preferred codon usage is advantageous for translational efficiency to increase protein expression. Finally, batch fermentation was applied, and the extracellular coα-CGTase enzyme activity was 326?% that of wtα-CGTase. The results suggest that codon optimization is a reasonable strategy to improve the yield of α-CGTase for industrial application.     

AmyM,a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. Strain EGB

A novel α-amylase, AmyM, was purified from the culture supernatant of Corallococcus sp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of Corallococcus coralloides DSM 2259, the AmyM gene was identified and cloned into Escherichia coli. amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases. amyM was expressed in E. coli BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in E. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The K_m and V_max of recombinant AmyM for soluble starch were 6.61 mg ml⁻¹ and 44,301.5 μmol min⁻¹ mg⁻¹, respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.     

Expression of a metagenome-derived fumarate reductase from marine microorganisms and its characterization

A potential novel fumarate reductase gene designated frd1A was isolated by screening a marine metagenomic library through a sequence-based strategy. Sequence analyses indicated that Frd1A and other putative fumarate reductases were closely related. The putative fumarate reductase gene was subcloned into a pETBlue-2 vector and expressed in Escherichia coli Tuner(DE3)pLac? cells. The recombinant protein was purified to homogeneity. Functional characterization by high-performance liquid chromatography demonstrated that the recombinant Frd1A protein could catalyze the hydrogenation of fumarate to succinate acid. The Frd1A protein displayed an optimal activity at pH 7.0 and 28 °C, which could be stimulated by adding metal ions such as Zn²⁺ and Mg²⁺. The Frd1A enzyme showed a comparable affinity and catalytic efficiency under optimal reaction conditions: k _m?=0.227 mmol/L, v _max= 29.9 U/mg, and k _cat/k _m=5.44?×?10⁴ per mol/s. The identification of Frd1A protein underscores the potential of marine metagenome screening for novel biomolecules.     

Optimization of a pseudo-affinity process for penicillin acylase purification

A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampicillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is independent of pH between 5.5 and 8.8, in 100?mM phosphate containing 22% (w/v) ammonium sulphate. The desorption of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100?mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7?IU/mg prot). With this extract and an affinity gel of Sepharose 4B-CNBr derivatized with ampicillin (3.8?μmol/cm³?gel), a maximum activity capacity adsorbed of 20?IU/cm³?gel was obtained for initial values of activity and protein concentration of 1.7?IU/cm³ and 0.4?mg prot/cm³, respectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was characterized by a maximum purity of 34?IU/mg prot, corresponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electrophoresis presented 4 main stained bands with 2 corresponding to α and β subunits of the penicillin acylase with equivalent molecular weights of 27 and 63?kDa. No external diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8?μmol/cm³?gel) were observed for continuous adsorption processes performed at two different agitation speeds (120 and 400?rpm).     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

Development and evaluation of a dot blot assay for rapid determination of invasion-associated gene ibeA directly in fresh bacteria cultures of E. coli

The ibeA gene, one of the important invasion-associated genes in neonatal meningitis Escherichia coli (NMEC), has been recently detected in avian pathogenic E. coli (APEC). Thus, it is necessary to close monitor the possible contamination of the poultry farms and its products to people. Here, a dot blot method for detecting the ibeA gene in E. coli was developed and validated. For the present study, probe sequence was designed and optimized for the specificity of dot blot. A 342-bp conserved fragment of ibeA gene was selected and labeled with digoxigenin (DIG)-dUTP according to the manufacturer??s guidelines, which indicated that this probe hybridizes with ibeA. In our established method, the bacteria culture samples were directly spotted on the membrane, following simple lyses on the membrane. Hence, the extraction of genomic DNA is not required, which reduces the workload and shortens the time. Furthermore, this assay was very sensitive, which could detect as few as 2.5?×?10³?CFU bacteria. The diagnostic reliability of this dot blot was evaluated on 467 APEC bacteria samples by using PCR analysis. Both methods showed that the result was in complete concordance. The dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify invasion-associated genes ibeA, which could be applied for initial screening of a large number of clinical samples or direct detection of bacteria culture.     

Codon Optimisation Is Key for Pernisine Expression in Escherichia coli

Background

Pernisine is an extracellular serine protease from the hyperthermophilic Archaeon Aeropyrum pernix K1. Low yields from the natural host and expression problems in heterologous hosts have limited the potential applications of pernisine in industry.

Methodology/ Principal Findings

The challenges of pernisine overexpression in Escherichia coli were overcome by codon preference optimisation and de-novo DNA synthesis. The following forms of the pernisine gene were cloned into the pMCSGx series of vectors and expressed in E. coli cells: wild-type (pernisine^wt), codon-optimised (pernisine^co), and codon-optimised with a S355A mutation of a predicted active site (pernisine^S355Aco). The fusion-tagged pernisines were purified using fast protein liquid chromatography equipped with Ni²⁺ chelate and gel filtration chromatography columns. The identities of the resultant proteins were confirmed with N-terminal sequencing, tandem mass spectrometry analysis, and immunodetection. Pernisine^wt was not expressed in E. coli at detectable levels, while pernisine^co and pernisine^S355Aco were expressed and purified as 55-kDa proforms with yields of around 10 mg per litre E. coli culture. After heat activation of purified pernisine, the proteolytic activity of the mature pernisine^co was confirmed using zymography, at a molecular weight of 36 kDa, while the mutant pernisine^S355Aco remained inactive. Enzymatic performances of pernisine evaluated under different temperatures and pHs demonstrate that the optimal enzymatic activity of the recombinant pernisine is ca. 100°C and pH 7.0, respectively.

Conclusions/ Significance

These data demonstrate that codon optimisation is crucial for pernisine overexpression in E. coli, and that the proposed catalytic Ser355 has an important role in pernisine activity, but not in its activation process. Pernisine is activated by autoproteolytical cleavage of its N-terminal proregion. We have also confirmed that the recombinant pernisine retains the characteristics of native pernisine, as a calcium modulated thermostable serine protease.     

Detection of the mutagenic activity of lead chromate using a battery of microbial tests

The potential mutagenicity of the carcinogen lead chromate was tested by the following battery of microbial tests: the Escherichia coli PolA⁺/PolA⁻ survival test; the Salmonella/microsome His⁺ reversion assay; the E. coli Trp⁺ reversion test as a plate assay; the E. coli Gal⁺ forward mutation test; and the Saccharomyces cerevisiae assay for mitotic recombination. Lead chromate is mutagenic in Salmonella and in Saccharomyces and is thus identified as a microbial mutagen by this battery. Metabolic activation by rat liver homogenate (S9) is not required for the mutagenic activity of lead chromate. The most statistically significant, positive result is found with a supplementary assay, the E. coli fluctuation test. To determine whether the lead ion and/or the chromate ion were responsible for the mutagenicity observed, lead chloride and chromium trioxide (chromic acid) were also tested. In E. coli fluctuation tests, the ranges of maximal mutagenicity for chromium trioxide and lead chromate overlap at the concentration 10⁻⁵ M, whereas lead chloride shows no mutagenicity and little lethality at concentrations up to 10⁻³ M. Thus, it appears that the chromate ion is responsible for the mutagenicity of lead chromate.     

Engineered short branched-chain acyl-CoA synthesis in E. coli and acylation of chloramphenicol to branched-chain derivatives

Short branched-chain acyl-CoAs are important building blocks for a wide variety of pharmaceutically valuable natural products. Escherichia coli has been used as a heterologous host for the production of a variety of natural compounds for many years. In the current study, we engineered synthesis of isobutyryl-CoA and isovaleryl-CoA from glucose in E. coli by integration of the branched-chain α-keto acid dehydrogenase complex from Streptomyces avermitilis. In the presence of the chloramphenicol acetyltransferase (cat) gene, chloramphenicol was converted to both chloramphenicol-3-isobutyrate and chloramphenicol-3-isovalerate by the recombinant E. coli strains, which suggested successful synthesis of isobutyryl-CoA and isovaleryl-CoA. Furthermore, we improved the α-keto acid precursor supply by overexpressing the alsS gene from Bacillus subtilis and the ilvC and ilvD genes from E. coli and thus enhanced the synthesis of short branched-chain acyl-CoAs. By feeding 25 mg/L chloramphenicol, 2.96?±?0.06 mg/L chloramphenicol-3-isobutyrate and 3.94?±?0.06 mg/L chloramphenicol-3-isovalerate were generated by the engineered E. coli strain, which indicated efficient biosynthesis of short branched-chain acyl-CoAs. HPLC analysis showed that the most efficient E. coli strain produced 80.77?±?3.83 nmol/g wet weight isovaleryl-CoA. To our knowledge, this is the first report of production of short branched-chain acyl-CoAs in E. coli and opens a way to biosynthesize various valuable natural compounds based on these special building blocks from renewable carbon sources.     

Isolation and purification of expression product of Populus euphratica CPD (PeCPD) in Escherichia coli: A comparison of two different methods

An effective procedure for purifying the expression product of Populus euphratica constitutive photomorphogenesis and dwarf (PeCPD) gene in Escherichia coli had been developed. The product, which is a cytochrome P450 monooxygenase, participating in plant brassinosteroid (BR) biosynthesis was deduced. This method is based on the combination of differential centrifugation (DC) with preparative SDS-PAGE and below is named as DC-PSDS-PAGE. The target protein obtained by this method was apparently better in purity and yield than that obtained by Ni²⁺ chelate affinity chromatography (Ni²⁺-CAC). The target protein purity and yield was 95% and 37.897 mg/L of bacterial culture, respectively, in DC-PSDS-PAGE vs. 30% and 0.816 mg/L of bacterial culture in Ni²⁺-CAC. To the best of our knowledge, this procedure is reported for the first time. The reasons for selecting this method and its advantages are discussed in the paper. The purified protein can be used for raising antibody, which is necessary for in situ localization, and this will be a very important part of our subsequent experiments in studying the function of the target protein.     

Assay of ornithine aminotransferase by high-performance liquid chromatography

Ornithine aminotransferase has been measured previously with a spectrophotometric assay and with a radioactive assay. We report here an isocratic reverse phase high-performance liquid chromatography assay which measures Δ¹-pyrroline-5-car?ylic acid, the reaction product. This assay offers the advantages of sensitivity and convenience.     

Chemical constituents,antibacterial activity and mechanism of action of the essential oil from Cinnamomum cassia bark against four food-related bacteria

The majority of components of the essential oil from Cinnamomum cassia bark were identified by gas chromatography and mass spectrometry (GC-MS) in this study. The trans-cinnamaldehyde (68.52%) was found to be the major compound. The antibacterial activity of essential oil against four food-related bacteria was evaluated. The results showed it was stronger effect against Staphylococcus aureus with both the largest ZOI of 27.4 mm and the lowest minimum inhibitory concentration (MIC) of 2.5 mg/mL and minimum bactericidal concentration (MBC) of 5.0 mg/mL respectively. Postcontact effect (PCE) assay also confirmed the essential oil had a significant effect on the growth rate of surviving S. aureus and Escherichia coli. The mechanism against S. aureus and E. coli may be due to the increase in permeability of cell membranes, and the leakage of intracellular constituents based on cell permeability assay and electron microscopy observations.     

Synthesis and biological activity of [14a-3H]cryptopleurine

[14a-³H]Cryptopleurine was chemically synthesized from the perchlorate salt of 9,11,12,13,14,15-hexahydro-2,3,6-trimethoxyphenanthro(9,10-b)quinolizidinium by reduction with NaB³H₄. The [³H]cryptopleurine was recrystallized from acetone and further purified by chromatography through alumina using benzene as the eluting solvent. Both infrared and ultraviolet spectra of the labeled product were identical to those obtained using either the natural compound or the unlabeled synthetic compound. Thin-layer analysis on various solid supports using several different eluting solvents gave only one radioactive spot with a specific activity of 1438 Ci/mol, which in all cases cochromatographed with the natural sample. The [³H]cryptopleurine was also identical to the unlabeled compound in that it bound strongly to polyribosomes. 80 S ribosomes, and 40 S ribosomal subunits, all isolated from yeast. Binding was less strong using either 60 S ribosomal subunits or Escherichia coli ribosomes.