Agarose Affinity Chromatography Research Articles

The effects of complement-independent, autoantibody-induced apoptosis of platelets in immune thrombocytopenia (ITP).

Autoantibodies that cause platelet apoptosis may play a role in the development of immune thrombocytopenia (ITP), specifically antibodies that target GPIIbIIIa and GPIbα. Our research aims to compare the impact of the antigen specificity of antiplatelet antibodies on normal platelets under conditions that do not rely on complement. Using a modified monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay, we detected the levels of autoantibodies against specific platelet membrane glycoproteins (GPIIb/IIIa, GPIb/IX) in the plasma of 36 patients diagnosed with chronic ITP. IgG was isolated and purified using a protein A agarose affinity chromatography column, and their concentrations were measured using spectrophotometry. We obtained normal platelets and treated them with the purified IgG anti-GPIIb/IIIa and/or anti-GPIb/IX antibodies, as well as an IgG-free buffer and healthy control IgG. Flow cytometry was used to analyze markers of apoptosis, including phosphatidylserine (PS) exposure, mitochondrial inner membrane potential (ΔΨm), and platelet particle formation. Our results indicate that ITP patients with GPIb/IX-specific autoantibodies can induce platelet apoptosis and platelet particle formation through complement-independent pathways, which are not associated with platelet activation, while GPIIb/IIIa-specific autoantibodies did not have this effect. This suggests that specific autoantibodies may serve as a valuable predictive tool to identify patients who could potentially benefit from complement-inhibiting therapy in the future.

Maximization of uricase production in a column bioreactor through response surface methodology-based optimization

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme that is widely exploited for diagnostic and treatment purposes in medicine. This study focuses on producing recombinant uricase from E. coli BL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through the Plackett–Burman design from eight different variables and were further optimized by the central composite design of the response surface methodology (RSM). The selected variables included the inoculum size (%v/v), isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration (mM) and the initial pH of the culture medium. The activity of uricase, the final optical density at 600 nm wavelength (OD600) and the final pH were considered as the responses of this optimization and were modeled. As a result, activity of 5.84 U·ml−1 and a final OD600 of 3.42 were obtained at optimum conditions of 3% v/v inoculum size, an IPTG concentration of 0.54 mM and a pH of 6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165 ± 1.5 mg uricase was obtained from a 600 ml cell culture. The results of this study show that BCBs can be a highly effective option for large-scale uricase production.

Cloning, functional characterization and evaluating potential in metabolic engineering for lavender ( +)-bornyl diphosphate synthase.

We isolated and functionally characterized a new ( +)-bornyl diphosphate synthase (( +)-LiBPPS) from Lavandula x intermedia. The in planta functions of ( +)-LiBPPS were evaluated in sense and antisense transgenic plants. The monoterpene ( +)-borneol contributes scent and medicinal properties to some plants. It also is the immediate precursor to camphor, another important determinant of aroma and medicinal properties in many plants. ( +)-Borneol is generated through the dephosphorylation of bornyl diphosphate (BPP), which is itself derived from geranyl diphosphate (GPP) by the enzyme ( +)-bornyl diphosphate synthase (( +)-BPPS). In this study we isolated and functionally characterized a novel ( +)-BPPS cDNA from Lavandula x intermedia. The cDNA excluding its transit peptide was expressed in E. coli, and the corresponding recombinant protein was purified with Ni-NTA agarose affinity chromatography. The recombinant ( +)-LiBPPS catalyzed the conversion of GPP to BPP as a major product, and a few minor products. We also investigated the in planta role of ( +)-LiBPPS in terpenoid metabolism through its overexpression in sense and antisense orientations in stably transformed Lavandula latifolia plants. The overexpression of ( +)-LiBPPS in antisense resulted in reduced production of ( +)-borneol and camphor without compromising plant growth and development. As anticipated, the overexpression of the gene led to enhanced production of borneol and camphor, although growth and development were severely impaired in most transgenic lines strongly and ectopically expressing the ( +)-LiBPPS transgene in sense. Our results demonstrate that LiBPPS would be useful in studies aimed at the production of recombinant borneol and camphor in vitro, and in metabolic engineering efforts aimed at lowering borneol and camphor production in plants. However, overexpression in sense may require a targeted expression of the gene in glandular trichomes using a trichome-specific promoter.

Antiserum Preparation and Specific Detection of Banana RING-E3 Ubiquitin-Protein Ligase

According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni2+-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.

Expression of a β-glucosidase from Trichoderma reesei in Escherichia coli using a synthetic optimized gene and stability improvements by immobilization using magnetite nano-support

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low β-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized β-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The β-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the β-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, β-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

Cloning, Expression, Characterization, and Tissue Distribution of Cystatin C from Silver Carp (Hypophthalmichthys molitrix).

Cystatins are proteins, which inhibit cysteine proteases, such as papain. In this study, the 336-bp cystatin C gene (family II, HmCysC) of silver carp (Hypophthalmichthys molitrix) was cloned and expressed in Escherichia coli BL21 (DE3). HmCysC encodes the mature peptide of cystatin C (HmCystatin C), with 111 amino acids. A typical QXXXG motif was found in HmCystatin C and it formed a cluster with Cyprinus carpio and Danio rerio cystatin C in the phylogenetic tree. Quantitative real-time polymerase chain reaction analysis indicated that HmCysC was transcribed at different levels in five tested tissues of silver carp. Following purification with Ni2+- nitrilotriacetic acid agarose affinity chromatography, HmCystatin C displayed a molecular weight of 20 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified HmCystatin C had strong inhibitory effects toward the proteolytic activity of papain. Immunochemical staining with anti-HmCystatin C antibody showed that HmCystatin C was widely distributed in silver carp tissues. These results collectively demonstrated the properties of HmCystatin C, providing information for further studies of cystatins from fish organisms.

Investigation of Anti-proliferative and Anti-migratory Properties of Recombinant J2-C2 Against Tumor Cells

Cancer is a big public health problem in many parts of the world. A novel anti-tumor protein (J2-C2) was previously isolated from Arca inflata and it was reported that this protein had anti-proliferative effect on some human tumor cell lines such as A549, HepG2 and SPC-A-1. In this study, firstly, J2-C2 was produced by recombinant techniques in the Escherichia coli strain BL21 (DE3) pLysE and this protein was purified by Ni-NTA agarose affinity chromatography. Expressed recombinant J2-C2 was analyzed with SDS-PAGE. 75.5 mg ml-1 of J2-C2 was achieved from a 600 mL culture. Then using HT-29, MCF7 and PC3 cancer cell lines, we showed the effect of recombinant of J2-C2 on cell proliferation, migration and apoptosis in a cell specific manner. Cell viability was measured using MTT assay. Additionally, real-time-qPCR was applied to analyze the transcript levels of apoptosis related genes such as Bcl-2, Bax and p53. The 2–ΔΔCt method was performed to determine the relative changes in gene transcription. Moreover, scratch wound healing assay was performed to evaluate the effect of J2-C2 on cancer cell migration. Consequently, we found that recombinant J2-C2 did not have a significant effect on cell viabilities of MCF7, PC3 and HT29 in concentration-dependent manner. Furthermore, our results showed that recombinant J2-C2 declined HT29, MCF7 cell migration. However, we did not observe the same results for PC3 cancer cell line.

Deciphering of The Effect of Chemotherapeutic Agents on Human Glutathione S-Transferase Enzyme and MCF-7 Cell Line.

Cancer is the disease that causes the most death after cardiovascular diseases all over the world these days. Breast cancer is the most common type of cancer among women and ranks the second among cancer-related deaths after lung cancer. Chemotherapeutics act by killing cancer cells, preventing their spread and slowing their growth. Recent studies focus on the effects of chemotherapeutics on cancer cells and new chemotherapy approaches that targeting enzymes that catalyze important metabolic reactions in the cell. The aim of this study was to investigate the effects of chemotherapeutic agents, Tamoxifen and 5-FU, on MCF-7 cell line and human erythrocyte GST, an important enzyme of intracellular antioxidant metabolism. In this study, it was investigated that the effect of chemotherapeutic agents, Tamoxifen and 5-FU, on MCF-7 breast cancer cell line and performed ROS analyzes. In addition, it was purified glutathione S-transferase (GST), one of the important enzymes of intracellular antioxidant mechanism, from human erythrocytes by using ammonium sulfate precipitation and glutathione agarose affinity chromatography, and investigated in vitro effects of chemotherapeutic agents, 5 - FU and Tamoxifen, on the activity of this enzyme for the first time. it was determined that Tamoxifen and 5-FU inhibited cellular viability and 5-FU increased intracellular levels of ROS, whereas Tamoxifen reduced intracellular levels of ROS. In addition, human erythrocyte GST enzyme with 16.2 EU/mg specific activity was purified 265.97-fold with a yield of 35% using ammonium sulfate precipitation and glutathione agarose affinity chromatography. The purity of the enzyme was checked by the SDS-PAGE method. In vitro effects of chemotherapeutics, 5-FU and Tamoxifen, on GST activity purified from human erythrocytes were investigated. The results showed that 5-FU increased the activity of GST in the concentration range of 77 to 1155 μM and that Tamoxifen increased the activity of GST in the concentration range of 0.54 to 2.70 μM. In this study, the effects of tamoxifen and 5-FU chemotherapeutic agents on both MCF-7 cell line and human GST enzyme were examined together for the first time. Our study showed that chemotherapeutic agents (5-FU and Tamoxifen) inhibited cellular viability and Tamoxifen reduced intracellular levels of ROS whereas 5-FU increased intracellular levels of ROS. In addition, 5-FU and Tamoxifen were found to increase the activity of GST enzyme purified from the human erythrocyte.

Grass carp (Ctenopharyngodon idella) stefin A: Systematic research on its cloning, expression, characterization and tissue distribution

To fully understand the properties of piscine stefins (family I cystatins), the 294-bp stefinA gene from grass carp (Ctenopharyngodon idella, Ci) was cloned and expressed in E. coli BL21 (DE3). After purification by Ni2+-NTA agarose affinity chromatography, the CiStefin A protein was tested to have a molecular weight of 11.48 kDa and an isoelectric point of 8.7. The typical motif of the cystatins superfamily was characterized from CiStefin A (QVVQG). CiStefin A specifically inhibited the activity of papain and cathepsin B/L. The Ki value of CiStefin A against papain was 6.5 × 10−11 M. CiStefin A showed excellent heat and acid-base tolerance. StefinA gene transcription occurred in all tested tissues of grass carp, with the highest level in the hepatopancreas. Immunolocalization staining with an anti-CiStefinA antibody revealed the CiStefinA protein distribution in all tested tissues at various levels. Overall, these results clarified the physical and biochemical properties of grass carp stefin A.

The interaction methylene blue and glutathione-S-transferase purified from human erythrocytes

It is known that textile dyes have various risks on human health. Glutathione-S-transferase enzymes play a critical role in the detoxification of xenobiotics in living systems. This study aimed to examine the interaction of methylene blue with human erythrocyte glutathione-S-transferase purified in one step. Human erythrocyte glutathione-S-transferase was purified with approximately 750-fold purification and 30% efficiency by glutathione agarose affinity chromatography. The results showed that the enzyme was inhibited by methylene blue with an IC50 value of 1.40 mmol/L. The Ki constant of methylene blue was 1.17 mmol/L. The Lineweaver–Burk graph of the methylene blue showed that the type of inhibition was compatible with mixed type inhibition. A new third spot was also detected by thin-layer chromatography. Furthermore, the cytotoxicity of methylene blue on human erythrocytes was evaluated and it was found that the haemolysis per cent of methylene blue on erythrocytes was approximately 14%.

From in vitro to in silico: Modeling and recombinant production of DT-Diaphorase enzyme

DT-Diaphorase (DTD) belonging to the oxidoreductase family, is among the most important enzymes and is of great significance in present-day biotechnology. Also, it has potential applications in glucose and pyruvate biosensors. Another important role of the DTD enzyme is in the detection of Phenylketonuria disease. According to the above demands, at first, we tried to study molecular cloning and production of recombinant DTD in E. coli BL21 strain. We have successfully cloned, expressed, and purified functionally active diaphorase. The amount of enzyme was increased in 10-h using IPTG induction, and the recombinant protein was purified by Ni–NTA agarose affinity chromatography. After that, the kinetic and thermodynamic parameters of the enzyme, optimum temperature and pH were also investigated to find more in-depth information. In the end, to represent the connections between the structures and function of this enzyme, the molecular dynamics simulations have been considered at two temperatures in which DTD had maximum and minimum activity (310 and 293 K, respectively). The results of MD simulations indicated that the interaction between NADH with phenylalanine 232 residue at 310 K is more severe than other residues. So, to investigate the interaction details of NADH/PHE 232 the DFT calculations were done.

Cloning, Purification, and Characterization of the Catalytic C-Terminal Domain of the Human 3-Hydroxy-3-methyl glutaryl-CoA Reductase: An Effective, Fast, and Easy Method for Testing Hypocholesterolemic Compounds.

3-hydroxy-3-methyl glutaryl-CoA reductase, also known as HMGR, plays a crucial role in regulating cholesterol biosynthesis and represents the main pharmacological target of statins. In mammals, this enzyme localizes to the endoplasmic reticulum membrane. HMGR includes different regions, an integral N-terminal domain connected by a linker-region to a cytosolic C-terminal domain, the latter being responsible for enzymatic activity. The aim of this work was to design a simple strategy for cloning, expression, and purification of the catalytic C-terminal domain of the human HMGR (cf-HMGR), in order to spectrophotometrically test its enzymatic activity. The recombinant cf-HMGR protein was heterologously expressed in Escherichia coli, purified by Ni+-agarose affinity chromatography and reconstituted in its active form. MALDI mass spectrometry was adopted to monitor purification procedure as a technique orthogonal to the classical Western blot analysis. Protein identity was validated by MS and MS/MS analysis, confirming about 82% of the recombinant sequence. The specific activity of the purified and dialyzed cf-HMGR preparation was enriched about 85-fold with respect to the supernatant obtained from cell lysate. The effective, cheap, and easy method here described could be useful for screening statin-like molecules, so simplifying the search for new drugs with hypocholesterolemic effects.

Soluble expression and purification of high-bioactivity recombinant human bone morphogenetic protein-2 by codon optimisation in Escherichia coli.

We developed a simple method of preparing recombinant human bone morphogenetic protein-2 (rhBMP-2) with high biological activity. This rhBMP-2 was overproduced in Escherichia coli as a fusion protein with thioredoxin 6xHis-tag at its amino terminus. The cDNA fragment of human bone morphogenetic protein-2 (hBMP-2) fused to the secretion signal of alkaline phosphatase (PhoA) was expressed under T7 promoter in E. coli. After DNA sequence confirmation, the recombinant vector pETpho-bmp2 was transformed into E. coli BL21 (DE3). rhBMP-2 was produced by the recombinant strain pETpho-bmp2/BL21 (DE3) in a soluble form with an yield of 6.2mg/L culture. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) results showed that the molecular weight of the product was approximately 28 kD. Moreover, rhBMP-2 was secreted as a dimer with a natural structure. rhBMP-2, purified by Ni Nitrilotriacetic acid Agarose (Ni-NTA) affinity chromatography, was used to examine osteosarcoma MG-63 cells and assay the alkaline phosphatase (ALP) activity. Results showed that rhBMP-2 induced MG-63 cell differentiation. When the final concentration was 500ng/mL, the effect was more remarkable and ALP activity reached 525% compared with that of the control group.

Mycoplasma pneumoniae induces allergy by producing P1-specific immunoglobulin E

BackgroundOur previous study found that most Mycoplasma pneumoniae (MP) pneumonia (MPP)patients had elevated serum total immunoglobulin E (IgE) levels. ObjectiveTo determine components of MP that can cause an IgE increase in children, and to clarify its specific mechanism. MethodsThe components of MP cells were isolated by serum IgE from patients with MP pneumonia. These components obtained through the prokaryotic expression were used as allergens to detect the proportion of allergen-specific IgE produced in MPP patients, and the clinical characteristics and related immune parameters of these patients who produced this allergen-specific IgE were also analyzed. In addition, a cell experiment was used to verify the biological effect of these components in vitro. ResultsP1-specific IgE was detected in serum of MPP children. An approximately 24-kDa polypeptide of P1 protein was obtained through prokaryotic expression purified by nickel agarose affinity chromatography. Approximately 9.2% of MPP patients produced IgE against this polypeptide of P1 protein, which was more likely to be produced in MPP patients with no history of allergies or family history of allergy-related diseases. P1-specific IgE-positive MPP patients had more severe clinical symptoms, with excessive secretion of interleukin (IL)-4 and IL-5 and overdifferentiation of Th0 cells into Th2 cells. Tests also demonstrated that the P1 protein stimulated excessive secretion of IL-4 and IL-5 in peripheral blood mononuclear cells from the peripheral blood of healthy donors. ConclusionMycoplasma pneumoniae is not only an infectious agent but also an allergen for certain individuals. The P1 protein of MP can induce the production of P1-specific IgE.

Purification of glutathione S-transferase enzyme from quail liver tissue and inhibition effects of (3aR,4S,7R,7aS)-2-(4-((E)-3-(aryl)acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione derivatives on the enzyme activity.

The use of quail meat and eggs has made this animal important in recent years, with its low cost and high yields. Glutathione S-transferases (GST, E.C.2.5.1.18) are an important enzyme family, which play a critical role in detoxification system. In our study, GST was purified from quail liver tissue with 47.88-fold purification and 12.33% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by SDS-PAGE method and showed a single band. In addition, inhibition effects of (3aR,4S,7R,7aS)-2-(4-((E)-3-(aryl)acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7methanoisoindole-1,3(2H)-dion derivatives (1a-g) were investigated on the enzyme activity. The inhibition parameters (IC50 and Ki values) were calculated for these compounds. IC50 values of these derivatives (1a-e) were found as 23.00, 15.75, 115.50, 10.00, and 28.75μM, respectively. Ki values of these derivatives (1a-e) were calculated in the range of 3.04±0.50 to 131.50±32.50μM. However, for f and g compounds, the inhibition effects on the enzyme were not found.

Heterologous Expression and Isolation of Influenza A Virus Nuclear Export Protein NEP.

Influenza A virus nuclear export protein NEP (NS2, 14.4kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N- or C-terminal (His)6-tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

Inhibition of human erythrocyte glutathione S-transferase by some flavonoid derivatives

Glutathione transferase is one of the important enzymes for xenobiotic metabolism. Glutathione transferases catalyze the conjugation of various electrophilic endogenous and exogenous xenobiotics with γ-glutamyl-cysteinyl-glycine (GSH). GST activity can be changed by natural plant products. The present study’s goal is to examine the interaction of human erythrocyte GST with the natural plant compounds baicalin, baicalein, phloridzin, and phloretin. First, GST was purified from human erythrocyte with 1654-fold purification and 19.27% recovery by glutathione agarose affinity chromatography. The purity of the enzyme was checked by SDS-PAGE, showing single band, because it had a homodimer structure. Baicalin, baicalein, phloridzin, and phloretin flavonoids were shown to inhibit human erythrocyte GST with the IC50 values of 28.75, 57.50, 769.10, and 99.02 μM, respectively. The Ki constants for baicalin, baicalein, phloridzin, and phloretin flavonoids were 14.50 ± 0.71, 24.33 ± 2.08, 762.50 ± 85.97, and 86.49 ± 1.11 μM, respectively. According to these results, baicalin is the best inhibitor for human erythrocyte GST.

Recombinant production of Aspergillus Flavus uricase and investigation of its thermal stability in the presence of raffinose and lactose.

Aspergillus flavus uricase (Rasburicase) with a molecular mass of 135kDa is currently used for the treatment of gout and hyperuricemia occurring in tumor lysis syndrome. To characterize the effects of raffinose and lactose osmolytes on the uricase structure, its coding sequence was cloned, expressed in E. coli BL21, and purified by Ni-NTA agarose affinity chromatography. Thermal inactivation studies at 40°C showed that nearly 15% of UOX activity was preserved, while the presence of raffinose and lactose reduced its activity to 35 and 45% of its original activity, respectively. Investigation of UOX thermal stability at 40°C in the course of time showed that the enzyme relatively lost almost 60% of its original activity after 40min, whereas more than 50% of UOX activity is preserved in the presence of lactose. Estimation of thermal inactivation rate constant, k in, showed that the UOX k in and UOX k in in the presence of raffinose was unchanged (0.018min-1), whereas for the presence of lactose, it was 0.015min-1. Half-life and T m analysis showed that UOX half-life is almost 38min and addition of raffinose did not change the half-life, whereas the presence of lactose had remarkable impact on UOX half-life (46min). The presence of raffinose increased UOX T m to a lesser extent, whereas lactose notably enhanced the T m from 27 to 37°C. Overall, our findings show that lactose has protective effects on UOX stability, while for raffinose, it is relatively compromised.

Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia.

A methyl jasmonate responsive 3-carene synthase (Li3CARS) gene was isolated from Lavandula x intermedia and functionally characterized in vitro. Lavenders produce essential oils consisting mainly of monoterpenes, including the potent antimicrobial and insecticidal monoterpene 3-carene. In this study we isolated and functionally characterized a leaf-specific, methyl jasmonate (MeJA)-responsive monoterpene synthase (Li3CARS) from Lavandula x intermedia. The ORF excluding transit peptides encoded a 64.9kDa protein that was expressed in E. coli, and purified with Ni-NTA agarose affinity chromatography. The recombinant Li3CARS converted GPP into 3-carene as the major product, with K m and k cat of 3.69 ± 1.17µM and 2.01s-1 respectively. Li3CARS also accepted NPP as a substrate to produce multiple products including a small amount of 3-carene. The catalytic efficiency of Li3CARS to produce 3-carene was over tenfold higher for GPP (k cat /K m = 0.56µM-1s-1) than NPP (k cat /K m = 0.044µM-1s-1). Production of distinct end product profiles from different substrates (GPP versus NPP) by Li3CARS indicates that monoterpene metabolism may be controlled in part through substrate availability. Li3CARS transcripts were found to be highly abundant in leaves (16-fold) as compared to flower tissues. The transcriptional activity of Li3CARS correlated with 3-carene production, and was up-regulated (1.18- to 3.8-fold) with MeJA 8-72h post-treatment. The results suggest that Li3CARS may have a defensive role in Lavandula.

Production of biologically active recombinant goose FSH in a single chain form with a CTP linker sequence.

FSH is a glycoprotein hormone secreted by the pituitary gland that is essential for gonadal development and reproductive function. In avian reproduction study, especially in avian reproduction hormone study, it is hindered by the lack of biologically active FSH. In order to overcome this shortcoming, we prepared recombinant goose FSH as a single chain molecule and tested its biological activities in the present study. Coding sequences for mature peptides of goose FSH α and β subunits were amplified from goose pituitary cDNA. A chimeric gene containing α and β subunit sequences linked by the hCG carboxyl terminal peptide coding sequence was constructed. The recombinant gene was inserted into the pcDNA3.1-Fc eukaryotic expression vector to form pcDNA-Fc-gFSHβ-CTP-α and then transfected into 293-F cells. A recombinant, single chain goose FSH was expressed and verified by SDS-PAGE and western blot analysis, and was purified using Protein A agarose affinity and gel filtration chromatography. Biological activity analysis results showed that the recombinant, chimeric goose FSH possesses the function of stimulating estradiol secretion and cell proliferation, in cultured chicken granulosa cells. These results indicated that bioactive, recombinant goose FSH has been successfully prepared in vitro. The recombinant goose FSH will have the potential of being used as a research tool for studying avian reproductive activities, and as a standard for developing avian FSH bioassays.