Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein

Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

Purification and Characterization of Flammulin,a Basic Protein with Anti-tumor Activities from Flammulina velutipes

Aim To purify and characterize flammulin, a basic protein with anti-tumoractivities. Methods Ammonium sulfate, ethanol fractionation and column chromatography were used forseparation and purification. Electrophoretic analysis, amino acid analysis, and MS of flammulin werecarried out. Results Flammulin was purified to electrophoretic homogeneity and crystallized. With amolecular mass of 19891.13 Da, pI 8.9, λ_(max) = 276 - 278 nm, λ_(min) = 250 nm, flammulin wascharacterized by its lack of methionine. Fingerprint mapping of flammulin was determined by MALDI-MSfollowing in-gel protease digestion; no close matches were identified. Conclusion Flammulin waspurified to electrophoretic homogeneity, and its characteristics are discussed for the first time.

FIBRINOLYTIC PROTEASES FROM THE GUT OF LARVAE OF FLESH FLY, BOETTCHERISCA PEREGRINA (DIPTERA: SARCOPHAGAE): PURIFICATION AND CHARACTERIZATION

Three fibrinolytic proteases, which were designated as BPGFP1, BPGFP2 and BPGFP3 individually, were purified from the gut extract of larvae of Boettcherisca peregrina fed on artificial diet containing fibrin rich pig blood coagulated block. BPGFP1 consists of two subunits with MW 32 000 and 30 000. Both BPGFP2 and BPGFP3 are monomer with MW 40 000 and 28 000, respectively. These three proteases are similar in substrate and inhibitor specificity. All of them possess high activities against fibrinolytic protease specific substrates such as fibrin, Chromzym P, Chromzym UK and S 2288. They also strongly hydrolyze trypsin specific substrates Bz Phe Val Arg NA, cBz Gly Pro Arg NA, Bz Pro Phe Arg NA and Bz Val Gly Arg NA. PMSF, STI, LBTI and SBBI can inhibit activity of these proteases. Activities of these three fibrinolytic proteases were found to be maximal at alkaline range of pH 9.0～10.0.

Purification and Characterization of a Cathepsin B-Like Proteinase from Eggs of Antheraea Pernyi

A cathepsin B-like proteinase was purified from eggs of Antheraea pernyi by 5 steps of purification. The molecular mass of the proteinase are estimated to be 47 kDa by using SDS-polyacrylamide gel electro-phoresis. The activity of the purified proteinase were strongly inhibited by E-64 and Leupeptin. The optimum pH is 3.5 as determined by using the bovine hemo-globin as substrate. The activity and quantity of the proteinase during embryo development were studied. The results suggested that the proteolytic activities during embryo development at least partially came from this proteinase.

Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Lipoxygenase （LOX, EC1.13.11.12） is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a（＋） and transformed into Escherichia coil BL21 （DE3）. Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside （IPTG） and the purified recombinant protein was obtained by His.Bind Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 （acetate buffer） and the optimum temperature was 30℃ for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk

A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase （GAD） activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps： 30%-90% saturation （NH4）2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel filtration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The purified GAD had a half-life of 50rain at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22.5＇-pyridoxal phosphate （PLP） had little effect on the regulation of its activity.

Purification and Characterization of a New Thermostable κ-CarrageenasefromtheMarineBacterium Pseudoalteromonas sp. QY203

A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KC1 increased its activity slightly. The divalent and trivalent metal ions including Cu^2＋, Ni^2＋, Zn^2＋, Mn^2＋, Al^3＋ and Fe^3＋ significantly inhibited its activity, while Mg^2＋ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.

Purification and Characterization of a Proteolytic Enzyme from Fig Latex

Ficin is an important component of plants in Ficus family such as fig latex. It is of special significance in medicine and industry because it exhibits activity throughout a wide range of temperature and pH values. In this work, we purified a component of ficin from the latex homogeneity of Shandong fig trees, and the properties of the purified ficin were studied. The current findings revealed that heavy metal ions were able to inhibit ficin, while DTT, L-cysteine, and β-ME were found to promote ficin activity. It was also observed that the half life of ficin at 65 °C was longer than 1 h and the Michaelis constant（Km） for casein hydrolyzation was determined to be 1.56 mg/mL. Our study shows that this purified ficin is a cysteine protease.

Purification and characterization of alkaline chitinase from Paenibacillus pasadenensis CS0611

An extracellular chitinase produced by Paenibacillus pasadenensis CS0611was purified by ammoniumsulfate precipitation,HiTrap DEAE FF and HiLoad26/600Superdex200pg column chromatography.The apparent molecular mass determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis was69kDa.The optimum pH and optimum temperature of the chitinase were5.0and50°C,respectively.The enzyme showed high stability at alkaline pH values and temperaturesbelow40°C.Additionally,the metal ions Mn2+,Mg2+,and Co2+inhibited activity of the chitinase.Thechitinase was active on colloidal chitin with an apparent Km of4.41mg/mL and Vmax of1.08mg/min.Substrate spectrum analysis indicated that the chitinase reacted preferentially with the glucosidicbond between GlcNAc‐GlcNAc.The enzymatic hydrolysate was analyzed by high‐performance liquidchromatography and thin layer chromatography,and clearly showed that a subunit of(GlcNAc)2was the main hydrolysis product.

Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility

This paper reports the purification and characterization of kinetic parameters of cellulase produced from Trichoderma viride under still culture solid state fermentation technique using cheap and an easily available agricultural waste material, wheat straw as growth supported substrate. Trichoderma viride was cultured in fermentation medium of wheat straw under some previously optimized growth conditions and maximum activity of 398±2.43U/mL obtained after stipulated fermentation time period. Cellulase was purified 2.33 fold with specific activity of 105U/mg in comparison to crude enzyme extract using ammonium sulfate precipitation, dialysis and Sephadex-G-100 column chromatography. The enzyme was shown to have a relative low molecular weight of 58kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis. The purified enzyme displayed 6.5 and 55oC as an optimum pH and temperature respectively. Using carboxymethyl cellulose as substrate, the enzyme showed maximum activity (Vmax) of 148U/mL with its corresponding KM value of 68μM. Among activators/inhibitors SDS, EDTA, and Hg2+ showed inhibitory effect on purified cellulase whereas, the enzyme activated by Co2+ and Mn2+ at a concentration of 1mM. The purified cellulase was compatible with four local detergent brands with up to 20 days of shelf life at room temperature suggesting its potential as a detergent additive for improved washing therefore, it is concluded that it may be potentially useful for industrial purposes especially for detergent and laundry industry.

Purification and characterization of novel κ-carrageenase from marine Tamlana sp.HC4

We isolated a bacterial strain （HC4） that is able to degrade k-carrageenan from a live specimen of the red alga Hyalosiphonia caespitosa. With 16S rRNA gene sequencing, we identified the strain as Tamlana sp., and then purified an extracellular K-carrageenase from a culture of Tamlana sp. HC4 by ammonium sulfate precipitation, Sephadex G-200 gel filtration chromatography, and DE-cellulose 52 anion-exchange chromatography. The purified enzyme yields a single band on SDS-PAGE with a molecular mass of 66.4 kDa. The optimal pH and temperature for κ-carrageenase activity are at 8.0 and 30~C, respectively. The enzyme is stable over the range ofpH 7.2-8.6 below 45℃. The enzyme activity is strongly inhibited by Zn2＋ and Cu2＋ at 1 mmol/L. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics with the Michaelis constant （Kin） at 7.63 mg/ml. Analysis of the degradation products of the κ-carrageenase by ESI-MS and 13C-NMR spectroscopy indicates that the enzyme degrades κ-carrageenan down to the level ofκ-neocarrabiose sulfate.

Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50oC,respectively.The protease activity is largely enhanced by Ca2 +,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml -1and 21.32μg·min -1·ml -1, respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.

Purification and characterization of manganese peroxidases from native and mutant Trametes versicolor IBL-04

Extracellular manganese peroxidases （MnPs） produced by native and mutant strains of Trametes versicolor IBL‐04 （EB‐60, EMS‐90） were purified by ammonium sulphate precipitation and dialysis, followed by ion‐exchange and gel‐permeation chromatography. The purified enzymes elucidated a single band in the 43‐kDa region on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. The optimum pH and temperature of the purified enzymes were found to be 5.0 and 40 °C, respec‐tively. Mutant strain MnPs exhibited a broader active pH range and higher thermal stability than native MnP. Purified MnPs from selected mutants showed almost identical properties to native MnP in electrophoresis, steady‐state kinetics, and metal ion and endocrine‐disrupting compound （EDC） degradation efficiency. Although the fastest reaction rates occurred with Mn2＋, MnPs displayed the highest affinity for ABTS, methoxyhydroquinone, 4‐aminophenol and reactive dyes. MnP activity was significantly enhanced by Mn2＋and Cu2＋, and inhibited in the presence of Zn2＋, Fe2＋, ethylene‐diaminetetraacetic acid and cysteine to various extents, with Hg2＋ as the most potent inhibitory agent. MnPs from all sources efficiently catalyzed the degradation of the EDCs, nonylphenol and triclosan, removing over 80%after 3 h of treatment, which was further increased up to 90%in the presence of MnP‐mediator system. The properties of T. versicolor MnPs, such as high pH and ther‐mal stability, as well as unique Michaelis‐Menten kinetic parameters and high EDC elimination effi‐ciency, render them promising candidates for industrial exploitation.

Purification and Characterization of a Novel Hydrolase That Can Specifically Degrade the Polysaccharide Isolated from Green Seaweed Ulva prolifera

The extracellular polysaccharide hydrolase-producing strain EP-1 was isolated from seawater and identified as Paenibacillus pabuli. Furthermore, a homogeneous extracellular polysaccharide hydrolase from Paenibacillus pabuli EP-1 was purified by combining ion-exchange chromatography and size exclusion chromatography with a purification fold of 90.69 and recovery of 16.23%. Characterization of the purified polysaccharide hydrolase revealed a molecular mass of 38 k Da and optimum activity at 45℃ and pH 6.0. The polysaccharide hydrolase maintained its stability within a wide range of pH(3.0–12.0) and thermal stability when the temperature was below 50℃. The presence of Hg^(2+), Fe^(2+), Mn^(2+), Co^(2+) and SDS notably decreased hydrolase activity, and organic solvents such as formaldehyde, acetone, DMF and acetonitrile completely inhibited hydrolase activity. The purified hydrolase had no activity on agar, carrageenan, gellan gum, sodium alginate, or starch, but effectively hydrolyzed the polysaccharide from Ulva prolifera. The Km and Vmax values of this hydrolase were 43.84 mg m L^(-1) and 4.33 mg m L^(-1) min^(-1), respectively. The sequence analysis with quantitative time-of-flight mass spectrometry indicated that the hydrolase was an endoglucanase.

Production, Purification and Characterization of Inulinase from a Newly Isolated <i>Streptomyces</i>sp. CP01

Inulinase is an enzyme catalyzing the hydrolysis of inulin, a plant reserve polysaccharide, into fructoses and fructooligosaccharides which are widely used as food additives. Here we report inulinase from a newly isolated Streptomyces as in the past decade there have been very few reports on inulinases from Streptomyces, especially purification and characterization of these enzymes. Out of 371 Streptomyces isolates, Streptomyces sp. CP01 produced highest inulinase activity of 0.50 U/ml. The enzyme activity was increased to 1.60 U/ml when CP01 was cultivated under the optimal conditions which consisted of using basal medium (Czapek’s Dox) containing 1% (w/v) inulin extract from Jerusalem artichoke’s root tubers and 0.7% (w/v) tryptone at pH8, shaking at 200 rpm and 28℃ for 24 h. The enzyme was purified from culture filtrate to about 67-fold purity by (NH4)2SO4 precipitation followed by four consecutive column chromatography steps. The purified enzyme is a single peptide with approximate molecular mass of 73 kDa as analyzed by gel filtration and 70.8 kDa as assessed by SDS-PAGE. The enzyme is optimally active at 55℃ and pH 6.0, however it still possesses more than 80% of the maximal activity at pH ranging from 5.5 to 9.0. It is stable at temperature up to 50℃ and at broad range of pH from 5.0 to 9.0 for 30 min. Its Km and Vmax values for inulin were 2.34 mM and 440 μmolmin–1mg–1, respectively. This enzyme has potential for industrial application as it is active at moderately high temperature and wide range of pH.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

Purification and Characterization of Superoxide Dismutase(SOD) from Camellia Pollen

A superoxide dismutase（ SOD ） was purified to homogeneity from fresh camellia pollen by means of ammonium sulfate precipitation and column chromatography with DEAE-cellulose（ DE52 ）, Sephadex G-100 and phenyl sepharose^TM 6 Fast Flow columns. Its specific activity could reach to 4034 U/mg protein and it was determined to be Cu/ Zn-SOD according to its different sensitivities to different inhibitors. The molecular weight of the SOD and its subunit were 69500 and 34700, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）, which implicates that the SOD in camellia pollen is a dimmer composed of two identical subunits. The isoelectric point of the enzyme was determined to be 4. 1 by isoelectric focusing electrophoresis and the N-terminal amino acid was identified to be Gly by the DNS-Cl method. Its α-Helix was also calculated to be approximately 21.8% according to the circular dichroism（CD） spectra.

Purification and Characterization of Cyclic AMP-Binding Protein from Ganoderma lucidum

Cyclic AMP-binding protein was purified 30 fold from the mycelia of Ganoderma lucidum by the methods of ammonium sulfate precipitation, DEAE-cellulose, phospho-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration. The molecular mass of the purified protein is 34.5 kDa and 17 kDa by Sephacryl S-100 gel filtration and SDS-ployacrylamide gel electrophoresis, respectively. From these results it is suggested that the protein has a homometric dimmer structure. The pI of the purified protein is pH 8.2 by native isoelectric focusing gel. The half-life of the protein activity in 10% glycerol at 4 ℃ is 7 d in crude extract, but its half-life is only 3 d under purifying conditions. The optimal conditions of the protein activity are at 1 ℃ and pH 7.5. Its activity is increased 6 times by 1 mmol/L Zn^(2+) and is slightly inhibited by cGMP, Cu^(2+) and Mn^(2+).

Characterization of Crude Cellulase from Trichoderma reesei and Purification of Cellulase

The gel filtration was carried out for purification of cellulase. The influences of chromatographic parameters on the resolution were studied to determine the optimal conditions for purification. The purified endoglucanase was obtained by gel filtration by Superdex 75 prep grade with an activity recovery of 92.8% and the purification factor 4.2. The sample volume should be below 6% of the column bed volume and the column bed height L≥12.0 cm. The optimum catalysis temperature and pH for the enzyme were 55 ℃ and 4.5—5.0, respectively. The cellulase was stable at pH ranging from 4.0 to 6.0 and temperature below 60 ℃.

Purification and characterization of vitellin in mature ovary of marine crab Charybdis japonica

Vitellin of mature female marine crab Charybdis japonica was purified by high-performance liquid chromatography (HPLC, DEAE-cellulose-16 anion exchange column). The apparent molecular mass of the vitellin is 546 ku based on the data of native-PAGE. Under denatured condition (SDS-PAGE), it was found that vitellin was composed of four polypeptides each at 120, 100, 65, and 55 ku. One disulfide bond was detected in the binding of polypeptide subunits. The purified vitellin, contained 4.47% phosphor and 10.6% polysaccharides, and was identified as glyco-lipo-carotenoprotein, according to the PAGE staining data. The purified vitellin can be used as antigen to raise polyclonal antisera in further application.