Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase(ΔTC-PTP)——Immunohistochemical Study of ΔTC-PTP Expression in Non-small Cell Lung Carcinomas

This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(ΔTC-PTP) and to study immunohistochemically the expression of ΔTC-PTP in human non-small cell lung cancers. ΔTC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-ΔTC-PTP was expressed in E.coli Rosetta(DE3) host cells and purified. The enzymatic characteristics of ΔTC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant ΔTC-PTP. Rabbit polyclonal antibody against ΔTC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against ΔTC-PTP protein. ΔTC-PTP gene was correctly cloned, expressed, and purified. The recombinant ΔTC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of ΔTC-PTP(76.92%, 10/13) than adenocarcinoma(57.14%, 4/7) and normal lung tissue(20%, 1/5). This study represents the first demonstration that ΔTC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.     

Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase（△TC-PTP） —— Immunohistochemical Study of △TC-PTP Expression in Non-small Cell Lung Carcinomas

This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase（△TC-PTP） and to study immunohistochemically the expression of △TC-PTP in human non-small cell lung cancers. △TC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-△TC-PTP was expressed in E. coli Rosetta （ DE3 ） host cells and puri- fied. The enzymatic characteristics of △TC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant △TC-PTP. Rabbit polyclonal antibody against △TC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against △TC-PTP protein. △TC-PTP gene was correctly cloned, expressed, and purified. The recombinant △TC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of △TC-PTP （76. 92%, 10/13 ） than adenocarcinoma （57.14%, 4/7） and normal lung tissue（20%, 1/5 ）. This study represents the first demonstration that △TC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.     

Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-ΔSHP-1 Antibodies

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta（DE3） E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.     

Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-△SHP-1 Antibodies

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated △SHP-1) and the preparation of its polyelonal antibodies.A cDNA fragment encoding △SHP-1 was amplified by PCR and then cloned into the pT7 expression vector.The recombinant pT7-△SHP-1 plasmid was used to transform Rosetta(DE3) E.coll cells.△SHP-1 was distributed in the exclusion body of E.coll cell extracts and was purified through a two-column chromatographic procedure.The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns.It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases.To generate polyclonal anti-△SHP-1 antibodies,purified recombinant △SHP-1 was used to immunize a rabbit.The resultant anti-serum was subjected to purification on △SHP-1 antigen affinity chromatography.The purified polyclonal antibody displayed a high sensitivity and specificity toward △SHP-1.This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.     

Expression Optimization and Characterization of the Catalytic Domain of Human MT3-MMP

Introduction Matrixmetalloproteinases(MMPs)areafamilyof calciumandzincrequiringendoproteinasesthattogether candegradeallthemaincomponentsoftheextra cellu larmatrixandbasementmembranes[1].MMPsarein volvedinawiderangeofproteolyticevents,innormal andpatholog…     

Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPα.     

Characterization and Activity Determination of the Human Protein Phosphatase 2A Catalytic Subunit α Expressed in Insect Larvae

Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit ?? of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit ?? of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250???g per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94???mol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2?months at ?20?°C.     

Expression and Characterization of Human Heart Type Fatty Acid Binding Protein in Pichia Pastoris

Introduction Fattyacidbindingproteins(FABPs)werefirstly foundbyOckneretal.,in1972[1],anddescribedas beingatypeofhomologicalcytoplasmicproteinswith molecularmassesaround12—16kDa.FABPsdistrib utewidelyinintestinalmucosa,liver,myocardium,head,skeletalandoth…     

Prokaryotic Expression, Purification, Antibody Production of a NH2 -Terminal Fragment of mCLCA3 Protein and Analysis of mClCA3 Protein Expression in Asthmatic Mouse Lung

mCLCA3 is a member of calcium activated chloride channel(CACC)family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung.To study the protein structure and expression of mCLCA3 in asthmatic mouse lung,an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E.coli,purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated.Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen.Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3.The results demonstrate that the 90 kDa N-terminal peptide,neither the full-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.     

A Novel Fusion Protein Diphtheria Toxin–Stem Cell Factor (DT-SCF)—Purification and Characterization

Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1–387 amino acids of diphtheria toxin, His-Ala linker, 2–141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.     

Synthesis,Characterization and Catalytic Activity of Salen Co(Ⅲ)Cl in Alternating Copolymerization of CO2 and Propylene Oxide

The title complex Salen Co(Ⅲ)Cl(Salen = 6,6’-((1E,1’E)-(cyclohexane-1,2-diylbis(azaneylylidene))bis(methaneylylidene))bis(2,4-di-tert-butylphenol)) was synthesized and characterized by elemental analysis, IR spectroscopy, 1H NMR and UV-Vis. The complex can be used as catalyst for the propylene oxide(PO)/CO2 copolymerization in different conditions of reaction time, reaction temperature, carbon dioxide pressure and monomer concentration, and the optimum conditions for copolymerization were obtained.     

Total Synthesis of (±)-Nocardione A and (±)-Nocardione B, Two Cdc25B Tyrosine Phosphatase Inhibitors

Quinones are biologically important compounds, especially because of their cytotoxic and pharmacological action. The antitumor activities of the quinones, including naphthoquinones1, were revealed more than two decades ago when the National Cancer Institute published a report in which 1500 synthetic and nature quinones were screened. Some natural naphthoquinones were under development for antitumor agents2. The protein tyrosine phosphatases (PTPase) and dual phosphatases (DSPase) are key…     

Synthesis,Structural Characterization and Catalytic Activity of Copper(Ⅰ) Complexes with Triphenylphosphine and Pyridine Derivatives

Two new Cu(Ⅰ) complexes [CuCl(3-PyOH)(PPh_3)_2](1) and [Cu_2(μ-Cl)_2(4-Stpy)(Ph_3P)_3](2)(PyOH = hydroxypyridine; stpy = styrylpyridine) with triphenylphosphine and pyridine derivatives have been synthesized and characterized by elemental analysis and X-ray single-crystal diffraction. Compound 1 crystallizes in monoclinic, space group P2_1/c with a = 9.8945(7), b = 37.266(2), c = 10.9461(7) ?, β = 116.0750(10)°, V = 3625.3(4) ?~3, Z = 4, D_c = 1.350 Mg/cm~(-3), μ = 0.801 mm~(-1), F(000) = 1528, the final R = 0.0320 and w R = 0.0729 for 18568 observed reflections(I 2σ(I)), R(all data) = 0.0413, wR(all data) = 0.0769, completeness to theta of 25.01 is 99.9% and GOF = 1.037. Compound 2 crystallizes in monoclinic, space group P2_1/c with a = 11.290(3), b = 20.388(5), c = 24.092(6) ?, β = 102.028(4)°, V = 5424(2) ?~3, Z = 4, D_c = 1.428 Mg/cm~(-3), μ = 1.016 mm~(-1), F(000) = 2408, the final R = 0.0568 and w R = 0.1486 for 27644 observed reflections(I 2σ(I)), R(all data) = 0.0716, wR(all data) = 0.1592, completeness to theta of 25.01 is 99.9% and GOF = 1.056. Two new Cu(I) complexes have been utilized as catalysts for N-arylation of imidazole and both showed good catalytic activity.     

Total synthesis of natural and non-natural Δ(5,6)Δ(12,13)-jatrophane diterpenes and their evaluation as MDR modulators

We report the details of the total synthesis of natural and non-natural jatropha-5,12-dienes. The successful tactic for the assembly of the strained trans-bicyclo[10.3.0]pentadecane scaffold employed a B-alkyl Suzuki-Miyaura cross-coupling for the formation of the C5/C6 double bond and a ring-closing metathesis for the construction of the C12/C13 double bond. The key step of the synthesis of the cyclopentane fragment, an uncatalyzed intramolecular carbonyl-ene reaction, was studied computationally by DFT calculations. The members of the ensemble of synthetic natural and non-natural jatrophanes were subsequently examined as modulators for the ABCB1, ABCG2, and ABCC1 efflux proteins, which are associated with multidrug resistance in cancer chemotherapy.     

Hydrophobic Pocket Modification and Humanized Catalytic Antibodies with Glutathione Peroxidase Activity(Ⅰ)——Preparation and Characterization of Haptenes and Conjugates

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Characterization and in vitro Permeation Study of Poly(ϵ‐caprolactone) Nanocapsules Containing Hinokitiol

Nanocapsules containing hinokitiol (HKL) were prepared by an emulsion‐diffusion method. Poly(?‐caprolactone) (PCL), synthesized according to a ring‐ opening method, was used as a wall material, and HKL dissolved in octylsalicylate (OS), was used as a core material. Under a microscope, submicron‐sized microcapsules were observed. The apparent shape was either spherical or amorphous. According to the image analyzer analysis, the size ranges from tenths of a nanometer to one micron, and more than 50% in number were less than 300 nm. In a calorimetric study, HKL exhibited an endothermic peak around 52°C, but no peak for the melting of HKL was observed when it was encapsulated in microcapsules. This suggests HKL remained dissolved in the nanocapsule cores but did not exist as a solid crystalline. In an in vitro permeation study, the flux of HKL encapsulated in the nanocapsules was somewhat higher than that of HKL dissolved in either propylene glycol or ethanol. This is probably because that PCL is hydrophobic, so the nanocapsules would readily interact with skin lipid.     

Pentamethylcyclopentadienylruthenium(II) Complexes: Synthesis,Characterization and Catalytic Activity in Aryl–Aryl Coupling

Ruthenium(II) complexes of the type [Ru(PPh₃)( ⁵-C₅Me₅)L] have been synthesized by the reactions of [RuCl(PPh₃)₂( ⁵-C₅Me₅)] with Schiff bases having the (N, O) donor atoms. The Schiff bases used in this study were prepared by condensing the appropriate aniline with salicylaldehyde or 2-hydroxy-1-naphthaldehyde in a 1:2 molar ratio respectively. The complexes were characterized by analytical, spectral (i.r., electronic and ¹H-n.m.r.) data. The new complexes have been used as catalysts in aryl–aryl coupling reactions.     

Study on the Synthesis and Characterization of Novel Immobilized β-Cyclodextrin Polymer (Ⅰ)

A series of novel β-cyclodextrin polymers was synthesized by immobilization of β-cyclodextrin on the chloromethylated crosslinked polystyrene carriers. The synthetic conditions such as reaction time, temperature, molar feeding ratio of reactants, the degree of crosslinking of polystyrene and the catalysts used were studied in detail and the chemical and physical structures of the formed β-CDpolymers were characterized. Results show that the preparation method is simple and the amount of β-CD immobilized is high. As biomedical adsorbents, they were tested for removal of various endogenous and exogenous toxins such as phenols, aromatic amins, barbitals and unconjugated bilirubin. Results indicate that the adsorptión capacity for those toxins can be enhanced by the inclusion interaction among the β-CD, the substrate molecules and the β-CD polymers.     

Synthesis and Characterization of Biodegradable Poly(ϵ-caprolactone)-b-Poly(L-lactide) and Study on Their Electrospun Scaffolds

A series of multi-block copolymers, poly(L-lactide)-b-poly (?-caprolactone) (PLLA-b-PCL) were synthesized. The first step of the synthesis consisted of the transesterification between the PLLA and 1,4-Butanediol, followed by the copolymerization of PLLA-diols and PCL, using isophorone diisocyanate (IPDI) as a coupling agent. The synthesized polymers were characterized by Fourier transform infrared (FTIR) spectra, differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD). PLLA/PCL block copolymers were electrospun into ultrafine fibers. The morphology of the electrospun fibrous scaffolds were investigated by Scanning Electron Microscopy (SEM). Results showed that the morphology and diameter of the fibers were affected by the electrospinning solution concentrationan and different weight ratio of PLLA/PCL. These electrospun PLLA-b-PCL fibrous membranes exhibited good flexibility and deformability. In comparison with the electrospun PLLA membrane, the electrospun fibrous membranes of PLLA-b-PCL demonstrated an enhanced elongation with still high tensile strength and Young's modulus to be beneficial for tissue engineering scaffolds.