Purification and characterization of Moschatin, a novel type I ribosomeinactivating protein from the mature seeds of pumpkin （Cucurbita moschata）, and preparation of its immunotoxin against human melanoma cells

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of-29 kD. It is a rRNA Nglycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a ICs0 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.     

Construction of a novel fusion protein harboring mouse inter- feron γ and epidermal growth factor receptor binding domain and enhancement of its antitumor activity

A novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain was constructed with the method of genetic and protein engineering. The fusion protein kept complete antiviral activity with the titer of 108 IU per liter of culture. The EGF-RBD of the fusion protein exhibited competitive binding activity against 125I-mEGF for mEGF receptors on A431 cells. The fusion protein was shown to be more potent in in-hibiting the growth of cultured mouse breast carcinoma cells than interferon γ. Experimental data on mouse B16 malig-nant melanoma model indicated that the tumor weight of fusion protein-treated group was statistically significantly smaller than that of interferon γ-treated group. The work here provides a necessarily reliable clue for the upcoming clinical employment of a novel class of targeting interferons.     

Purification of full-length human Pregnane and Xenobiotic Receptor： polyclonal antibody preparation for immunological characterization

Pregnane and Xenobiotic Receptor （PXR; or Steroid and Xenobiotic Receptor, SXR）, a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body＇s homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21（DE3） cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.     

Isolation and Characterization of a Novel Chitosan-Binding Protein from Non-Heading Chinese Cabbage Leaves

To know the mechanism of ammonia assimilation in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino) leaves regulated by chitosan (CTS), a CTS-binding protein was isolated from non-heading Chinese cabbage leaves using the chitosan affinity chromatography approach and this CTS-binding protein was partially characterized. The profile of the 53.1 kDa purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was compared with the native molecular weight of 106.5 kDa, which indicated that the purified protein was a dimer with identical subunits. After isoelectric focusing, a band was obtained at pH 8.25. The agglutination test and periodic acid-Schiff staining further revealed that the protein was a glycoprotein with lectin activity. Moreover, the purified protein contained 17.4% (w/w) neutral carbohydrate and 82.56% (w/w) protein. The comparison of this protein and the 67 kDa CTS-binding protein isolated previously from Rubus culture tissue exhibited some differences in characterization. According to results of peptide mass fingerprinting analysis, the protein purified in the present study does not show any similarity with any protein in the protein data bank. Thus, it was deduced that the protein purified in the present study is a novel CTS-binding protein.     

Isolation of a cDNA encoding a protease from Perinereis aibuhitensis Grube

The cDNA encoding a protease of Perinereis aibuhitensis Grube （PPA） was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21（DE3） and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-β-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the partially purified MoFe protein (Av 1) preparation was obtained from Azotobacter vinelandii Lipmann OP by chromatography on DEAE-cellulose (DE52) and Sephacryl S-200 columns and analyzed by PAGE and matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Ay 1 band, the preparation was shown to have three other main bands that migrated slower than Avl. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase (PGI). In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaCl concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.     

Human Calprotectin： Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S 100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 （physiological pH） and 8.0 （similar to intestinal pH） was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose- dependent manner. The intrinsic fluorescence emission spectra of human calprotectin （50 ktg/ml） in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the m-helical content of the protein and an increase in [3- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.     

Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.     

A Novel Missense Mutation （L^296Q） in Cholesteryl Ester Transfer Protein Gene Related to Coronary Heart Disease

Cholesteryl ester transfer protein (CETP) is a key participant in the reverse transport of cholesterol from the peripheral tissues to the liver. To understand the role that CETP gene plays in the pathogenesis of coronary heart disease (CHD) , the promoter region, all 16 exons and adjacent intronic regions of CETP gene were screened for single nucleotide polymorphisms (SNPs) in 203 CHD patients and 209 controls by a combination of PCR, denaturing high performance liquid chromatography (DHPLC), molecular cloning, and DNA sequencing. A novel missense mutation in the CETP gene was identified. This mutation (L296Q) was a T-to-A conversion at codon 296 of exon 10 which replaced the codon for leucine (CTG) with the codon for glutamine (CAG). Association study revealed that L296Q mutation was associated with CHD with a significantly higher mutant allele frequency in the CHD patients than that in the controls (0. 160 vs. 0.091,x2= 9.014, P = 0.003), and that the odds ratio for the development of CHD was 1.83 for     

Identification of melanoma-specific peptide epitopes by HLA-A2.1-restricted cytotoxic T lymphocytes

HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes （CTLs） by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and aUogeneic HLA-A2. 1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.     

A novel ubiquitin carboxy1 terminal hydrolase is involved in toad oocyte maturation

P28，a 28kD protein from toad (Bufo bufo gargarizans)oocytes,was identified by using P13^suc1-agarose affinity chromatography.Sequence homology analysis of the full-length cDNA of P28(Gene Bank accession number:AF 314091)indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70%positives to encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1,and contains homological functional domains of UCH family.Anti-p28 monoclonal antibody,on injecting into the oocytes,could inhibit the progesterone-induced resumption of meiotic division in a dose-dependent manner.The recombinant protein P28 showed similar SDS/PAGE behaviors to the native one,and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases(UCHs).The results in this paper reveal that a novel protein,p28 ,exists in the toad oocytes,is a UCH Ll homolog,was engaged in the process of progesterone-induced oocyte maturation possibly through an involvement in protein turnover and degradation.     

The structure analysis and antigenicity study of the N protein of SARS-CoV

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with o     

Studies on solution NMR structure of brazzein——Secondary structure and molecular scaffold

Brazzein is a sweet-tasting protein isolated from the fruit of West African plant Pentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far and is highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300 K) is presented. The complete sequence specific assignments of the individual backbone and sideehain proton resonances were achieved using through-bond and through-space eonneetivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one α-helix (residues 21—29), one short 3_(10)-helix (residues 14—17), two strands of antiparallel β-sheet (residues 34—39, 44—50) and probably a third strand (residues 5—7) near the N-terminus. A comparative analysis found that brazzein shares a so-called 'eysteine-stabilized alpha-beta' (CSαβ) motif with scorpion neurotoxins, insect defensins and plant γ-thionins. The significance of this multi-function motif, the possible active sites an     

Construction of a recombinant human GM-CSF/MCAF fusion protein and study on its in vitro and in vivo antitumor effects

A novel cytokine fusion protein was constructed by fusing granulocyte macrophage colony stimulat-ing factor (GM-CSF) with monocyte chemotactic activating factor (MCAF), which acts as a factor directing effector cells (monocytes) to a target site. The recombinant human GM-CSF/MCAF fusion protein could sustain the growth of GM-CSF-dependent cell line TF1 and was chemotactic for monocytes. The in vitro antitumor effect showed that rhGM-CSF/MCAF could activate monocytes to inhibit the growth of several human tumor cell lines, including a promyelocyte leukemia cell line HL-60, a lung adenocarcinoma cell line A549, a hepatoma cell line SMMC-7721 and a melanoma cell line Bowes. Furthermore, the cytotoxicity of monocytes activated by rhGM-CSF/MCAF against HL-60 and A549 was greater than that activated by GM-CSF or MCAF alone, even greater than that activated by a combina-tion of GM-CSF and MCAF, suggesting that the fusion protein has synergistic or enhanced effects. The in vivo anti-tumor effect indicated that     

Protein photo-folding and quantum folding theory

The rates of protein folding with photon absorption or emission and the cross section of photon-protein inelastic scattering are calculated from quantum folding theory by use of a field-theoretical method.All protein photo-folding processes are compared with common protein folding without the interaction of photons(non-radiative folding).It is demonstrated that there exists a common factor(thermo-averaged overlap integral of the vibration wave function,TAOI) for protein folding and protein photo-folding.Based on this finding it is predicted that(i) the stimulated photo-folding rates and the photon-protein resonance Raman scattering sections show the same temperature dependence as protein folding;(ii) the spectral line of the electronic transition is broadened to a band that includes an abundant vibration spectrum without and with conformational transitions,and the width of each vibration spectral line is largely reduced.The particular form of the folding rate-temperature relation and the abundant spectral structure imply the existence of quantum tunneling between protein conformations in folding and photo-folding that demonstrates the quantum nature of the motion of the conformational-electronic system.     

Galectin-1-mediated biochemical controls of melanoma and glioma aggressive behavior

Gliomas and melanomas are associated with dismal prognosis because of their marked intrinsic resistance to proapoptotic stimuli,such as conventional chemotherapy and radiotherapy,as well as their ability to escape immune cell attacks.In addition,gliomas and melanomas display pronounced neoangiogenesis.Galectin-1 is a hypoxia-sensitive protein,which is abundantly secreted by glioma and melanoma cells,which displays marked proangiogenic effects.It also provides immune tolerogenic environments to melanoma and glioma cells through the killing of activated T cells that attack these tumor cells.Galectin-1 protects glioma and melanoma cells against cytotoxic insults(including chemotherapy and radiotherapy) through a direct role in the unfolded protein response.Altogether,these facts clearly point to galectin-1 as an important target to be combated in gliomas and melanomas in order to:(1) weaken the defenses of these two types of cancers against radiotherapy,chemotherapy and immunotherapy/vaccine therapy;and(2) reinforce antiangiogenic therapies.In the present article,we review the biochemical and molecular biology-related pathways controlled by galectin-1,which are actually beneficial for melanoma and glioma cells,and therefore detrimental for melanoma and glioma patients.