MCL-1 is encoded by myeloid cell leukemia-1 gene (mcl-1), which is one of the anti-apoptotic members of bcl-2 cell apoptotic gene superfamily. ChanSu is made of dorsal secretions of several Bufo species, commonly used in the prescriptions of traditional Chinese medicine for treating many diseases including cancer. To clarify if mcl-1 is expressed in the dorsal skin of B. gargarizans, the PCR (polymerase chain reaction) was performed with its dorsal skin first strand cDNA as the template and a pair of specific primers of mcl-1, and PCR products were cloned into the pGM-T vector. DNA sequencing indicated that the ORF length was 639 bp encoding 212 amino acid residues, and the homology of 44%-95% with the MCL-1 of several other animals. For the further studies on MCL-1 biological functions during the oncogenesis and preparation of its antibody, the prokaryotic expression construct of pET-28b-mcl-1 was prepared which was confirmed by DNA sequencing, and its recombinant protein expression (0.02% wet weight) in E. coli BL21 (DE3) strain was confirmed by SDS-PAGE and Western blotting.
Amino Acid Sequence ; Animals ; Bufonidae ; classification ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; metabolism ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; metabolism ; Open Reading Frames ; genetics ; Phylogeny ; Recombinant Proteins ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology ; Skin ; metabolism

Hespintor is an unknown function protein that was got from hepatoblastoma cell lines HepG2 by suppression subtractive hybridization technique (SSH), sequence analysis showed that the protein is a new member of secretory type of Kazal type serine protease inhibitor (Serpin) family, and has high homology with esophageal cancer related gene 2 (ECRG2). The coding sequence of Hespintor's Kazal domain was subcloned into prokaryotic expression vector pET-40b(+), then transformed into Rosetta (DE3). A recombinant protein about 42 kDa in the form of inclusion body was optimization expressed by inducing with 0.25 mmol/L IPTG, 30 degrees C for 5 h. and its specificity was confirmed via Western blotting. The recombinant protein was purified by metal chelate affinity chromatography (MCAC) and anion-exchange chromatography. The preliminary experimental result showed that the recombinant protein can inhibit trysin hydrolysis activity specifically. The result clearly demonstrated that Hespintor, as a novel member of Serpin, would be valuable in developing anti-tumor agents.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hep G2 Cells ; Humans ; Proteinase Inhibitory Proteins, Secretory ; Recombinant Proteins ; biosynthesis ; genetics ; Serine Peptidase Inhibitors, Kazal Type ; Serine Proteinase Inhibitors ; biosynthesis ; classification ; genetics

A repetitive DNA fragment, named P1, was amplified by PCR with the full-length Minor Ampullate Spidroin gene sequence of Araneus ventricosus as template. P1 was ligated with pPic3.5 and PKT expression vectors and transferred into GS115 and BL21(DE3) competence cells, respectively. SDS-PAGE and Western blot were used to analyze the recombinant his-tag fusion protein. With expressed in different expression systems, soluble P1 induced proteins could be obtained as the same size. Furthermore, the expression level and purification recovery efficiency were also higher in GS115 than that of BL21(DE3). Additionally, the expression level could be improved after optimizing the incubation and induction conditions of GS115. In this research, Pichia pastoris expression system is more suitable for the native repetitive Gly/Ala-rich spider spidroin gene sequence expression than Escherichia coli system. The data can help the native full-length MiSp gene expression and large-scale exploitation of recombinant of spider silk proteins.
Animals ; Escherichia coli ; genetics ; metabolism ; Fibroins ; biosynthesis ; classification ; genetics ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Spiders ; classification ; genetics ; metabolism

Muscovy ducks reovirus (DRV) is an important pathogen with a high mortality rate in Muscovy ducks, the researches in the test and the immunity were useful for the prevention and control of DRV infection. In this study, the S3 genes of the three Fujian DRVs were cloned by RT-PCR and sequencing technology. It was found that DRV-YH and YJL were close to avian reovirus (ARV) in the genetic distance, with high identities ranged from 94. 6% to 98. 9%, however, the identities of DRV-YB strain and reference ARV strains in the S3 gene were only 60.6% - 61.7%. The expression vector pET-30a-S3 harboring DRV YB strain S3 gene was constructed and transformed into E. coli BL21, and then the fusion sigmaB protein expression was induced with IPTG. The SDS-PAGE of the expressed products indicated that the fusion protein of approximately 42ku in molecular weight was expressed highly in inclusion body, and made up 67. 7% of the total proteins. The most efficient concentration of IPTG and inducing time were 0. 1 mM and 5h respectively, while the best temperature for expression was 37 degrees C. After purification with the Ni2+ affinity chromatography, the fusion sigmaB protein was 93% of the total proteins, and the purified protein amounted to 0. 86g/L. The Western blot analysis showed that the fusion aB protein was recognized specifically by the antiserum against DRV, confirming that the recombinant fusion protein had good immunoreactivity.
Amino Acid Sequence ; Animals ; Capsid Proteins ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Molecular Sequence Data ; Orthoreovirus, Avian ; chemistry ; classification ; genetics ; isolation & purification ; Phylogeny ; Poultry Diseases ; virology ; RNA-Binding Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Reoviridae Infections ; veterinary ; virology ; Sequence Analysis ; Sequence Homology, Amino Acid

To examine the genetic background of avian pathogenic Escherichia coli (APEC) that affects virulence of this microorganism, we characterized the virulence genes of 101 APEC strains isolated from infected chickens between 1985~2005. Serotypes were determined with available anti-sera and median lethal doses were determined in subcutaneously inoculated chicks. The virulence genes we tested included ones encoding type 1 fimbriae (fimC), iron uptake-related (iroN, irp2, iucD, and fyuA), toxins (lt, st, stx1, stx2, and vat), and other factors (tsh, hlyF, ompT, and iss). Twenty-eight strains were found to be O1 (2.0%), O18 (3.0%), O20 (1.0%), O78 (19.8%), and O115 (2.0%) serotypes. The iroN (100%) gene was observed most frequently followed by ompT (94.1%), fimC (90.1%), hlyF (87.1%), iss (78.2%), iucD (73.3%), tsh (61.4%), fyuA (44.6%), and irp2 (43.6%). The strains were negative for all toxin genes except for vat (10.9%). All the strains were classified into 27 molecular pathotypes (MPs). The MP25, MP19, and MP10 pathotypes possessing iroN-fimC-ompT-hlyF-iucD-tsh-iss-irp2-fyuA (22.8%), iroN-fimC-ompT-hlyF-iucD-tsh-iss (21.8%), and iroN-fimC-ompT-hlyF-iss (11.9%) genotypes, respectively, were predominant. Redundancy of iron uptake-related genes was clearly observed and some strains were associated with higher mortality than others. Therefore, strains with the predominant genotypes can be used for diagnosis and vaccine.
Animals ; Bacterial Proteins/genetics/metabolism ; Chickens ; Escherichia coli/*classification/genetics/*pathogenicity ; Escherichia coli Infections/epidemiology/microbiology/*veterinary ; Gene Expression Regulation, Bacterial/physiology ; Phylogeny ; Poultry Diseases/epidemiology/*microbiology ; Republic of Korea/epidemiology ; Virulence

L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.
Aldose-Ketose Isomerases ; biosynthesis ; genetics ; Biotransformation ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Galactose ; metabolism ; Genetic Vectors ; genetics ; Hexoses ; metabolism ; Pediococcus ; classification ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVESecreted proteins of Helicobacter pylori (H. pylori) interact with gastric epithelium cells and may contribute to cell damage. Considering the fact that HP0175 and hypothetical conserved protein HP1286 are included in the group and that HP0175 is a well-known apoptosis-induced factor, the present study aims to clarify whether HP1286 plays a role in bacterial pathogenicity or even functions as an apoptosis-induced factor in human stomach.

METHODSTwo genes encoding HP1286 and HP0175 were cloned into pET32a vector and expressed as recombinant proteins in Escherichia coli (E. coli) BL21. Signal peptide sequences were not included. The recombinant proteins were purified with His SpinTrap and desalted by using HiTrap Desalting. Immunoreactivity of the proteins was determined by Western blot. Human gastric epithelial cell AGS was challenged with these endotoxin-free proteins; and apoptosis of cells was assayed with the Cell Death ELISA kit.

RESULTSRecombinant proteins and their respective products whose N-terminal his-tag were removed with thrombin were recognized by serum from the patient infected with H. pylori. Apoptotic AGS cells challenged by HP1286 of H. pylori 26695 were four times more than untreated cells. In addition, apoptosis-induced ability of HP1286 of SS1 was not as strong as that of H. pylori 26695 strain.

CONCLUSIONHP1286 of H. pylori 26695 induces apoptosis of AGS cells in a time-dependent manner, however the apoptosis-induced ability of HP1286 may differ due to variations between different strains.

Amino Acid Sequence ; Apoptosis ; drug effects ; Bacterial Proteins ; metabolism ; pharmacology ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; metabolism ; Gene Expression Regulation, Bacterial ; Genetic Variation ; Helicobacter pylori ; classification ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Recombinant Proteins ; Stomach Neoplasms

In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between alpha-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.
Animals ; Bacterial Proteins/analysis ; Bacterial Toxins/analysis ; Cat Diseases/microbiology ; Cats ; Cystitis/*microbiology ; Dog Diseases/microbiology ; Dogs ; Escherichia coli Infections/complications/microbiology/*veterinary ; Escherichia coli Proteins/analysis ; Female ; Genetic Variation ; Hemolysin Proteins/analysis ; Italy ; Male ; Operon ; Phylogeny ; Polymerase Chain Reaction ; Pyelonephritis/*microbiology ; Uropathogenic Escherichia coli/classification/*genetics/i ; Virulence Factors/*genetics

OBJECTIVETo clone and characterize the DNA and cDNA sequences of phenylalanine ammonia-lyase gene (PAL) from Fagopyrum dibotrys, and investigate the biological activity of the obtained PAL.

METHODUsing homology cloning and RT-PCR techniques, the DNA and full-length cDNA sequences of PAL gene were amplified from F. dibotrys. The obtained sequences were analyzed by bioinformatics software. The ORF of PAL gene was cloned into expression vector pET-30b(+) and transformed into Escherichia coli BL21 (DE3) for expression the recombined protein. The catalytic activity of the recombined protein was determined by Spectrophotometer and thin layer chromatography (TLC) methods.

RESULTThe DNA sequence of PAL gene (designated as FdPAL, GenBank accession number: HM628904) was 2 583 bp in size, of which consisted two extrons and a single intron, and the full-length cDNA of FdPAL was 2 169 bp in size, which contained an ORF. The deduced protein of FdPAL contained 722 amino acids with calculated molecular weight (MW) of 78.31 kDa and an isoelectric point (pI) of 5.94. The SDS-PAGE results showed that the molecular weight of recombinant FdPAL protein was 75.37 kDa, which is consistent with the predictions. After 4 hours of induction, the enzymatic specific activity of FdPAL reached the summit, up to 4 386 nmol x g(-1) x min(-1). The reaction products were also identified by TLC, using L-Phe and trans-cinnamic acid as the internal standard.

CONCLUSIONThe PAL gene (both DNA sequence and full-length cDNA sequence) was cloned from F. dibotrys, and it has the same classic characters as other PALs in plants. The recombinant FdPAL was efficiently expressed in E. coli and had the activity for catalyzing the conversion from L-phenylalanine to cinnamic acid.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fagopyrum ; classification ; genetics ; Molecular Sequence Data ; Phenylalanine Ammonia-Lyase ; chemistry ; genetics ; metabolism ; Phylogeny ; Protein Conformation ; Recombinant Proteins ; chemistry ; genetics ; metabolism

To obtain the pure and soluble P51 antigen of HIV-1 strain CN54, we transformed the Escherichia. coli strain BL21 codonplus-RIL with recombinant plasmid pTHioHisA51 which carries a gene encoding the Polymerase (Pol) P51 antigen of HIV-1 CN54 formerly, and induced protein expression by IPTG. We purified the recombinant protein with Chelating Sepharose FF-Ni and DEAE-Sepharose FF column chromatography, then renatured the recombinant protein by dialyzation. Purified protein was identified by Western blotting. We labeled and coated antigen P51 in a dual-antigen sandwich system, and tested it with serum samples from HIV-infected individuals. The results showed that P51 was expressed as inclusion body, and represented about 50% of total cellular protein. After purification and renaturation, the purity of P51 was up to 95%. Western blotting and sandwich ELISA demonstrated that recombinant P51 had good anti-HIV antibody specificity and sensitivity. The results suggested that recombinant HIV-1 P51 can be prepared as diagnostic reagent, and provides valuable support for HIV-1 detection and vaccine research.
Escherichia coli ; genetics ; metabolism ; HIV Antibodies ; blood ; immunology ; HIV Infections ; immunology ; virology ; HIV Reverse Transcriptase ; biosynthesis ; genetics ; immunology ; HIV-1 ; classification ; immunology ; Humans ; Protein Renaturation ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Sensitivity and Specificity