OBJECTIVETo construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli).

METHODSHuman GSTA1, GSTP1 and GSTT1 gene whole length cDNAs were amplified by RT-PCR and then subcloned into pET-28a(+) vectors. The proteins were expressed in E. coli BL21(DE3). After purified by Ni2+ affinity chromatography, the enzymatic activities of GSTs were measured with 1-chloro-2,4 -dinitrobenzene (CDNB) as substrate.

RESULTSThe correct GSTA1, GSTP1 and GSTT1 genes were cloned. And soluble GSTA1, GSTT1, GSTP1 proteins were expressed in E.coli. After purification, GSTA1, GSTT1 and GSTP1 showed good enzymatic activities, which were 17.55, 0.02, 18.75 μmol·min-1·mg-1, respectively.

CONCLUSIONThe expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully.

DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Glutathione S-Transferase pi ; biosynthesis ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody. The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene.
Animals ; Antibodies ; immunology ; isolation & purification ; metabolism ; Antigens, Neoplasm ; biosynthesis ; immunology ; isolation & purification ; Cells, Cultured ; Escherichia coli ; metabolism ; Exosome Multienzyme Ribonuclease Complex ; biosynthesis ; immunology ; isolation & purification ; Genetic Vectors ; Glutathione Transferase ; biosynthesis ; isolation & purification ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; RNA-Binding Proteins ; biosynthesis ; immunology ; isolation & purification ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification

Antimicrobial peptides (AMPs) are of great significance in the field of food, feed and medicine due to their wide spectrum of antimicrobial activity and new mechanism of action different from conventional antibiotics. AMPs production from natural sources is usually limited, and chemical synthesis is not economically practical, especially for the production of long peptides. Therefore, heterologous expression of AMPs has been widely studied as an alternative, and fusion expression plays an important role in increasing production. The present review mainly focuses on the types and bioactivities of AMPs. In addition, the recent strategies to the most commonly used carrier proteins for fusion expression of AMPs and prospects for future research were also discussed.
Anti-Infective Agents ; metabolism ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Glutathione Transferase ; biosynthesis ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thioredoxins ; biosynthesis ; genetics

To investigate the function of CTCF and understand the pathogenesis of tumors better, we produced rabbit polyclonal antibody of human transcription factor CTCF protein and detected its expression in several kinds of human cancer cells and tissues. GST fusion protein of human CTCF-N domain was purified by GSTrap-FF affinity chromatography and was successfully expressed under induction of IPTG in E. coli BL21. Western blotting analysis demonstrated that the polyclonal antibody can recognize the endogenous CTCF from HepG2, MCF-7 and HeLa cells specifically. The produced antibodies can be used for gene expression regulation and tissue distribution study at protein level.
Animals ; Antibodies ; immunology ; metabolism ; CCCTC-Binding Factor ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; HeLa Cells ; Hep G2 Cells ; Humans ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo express and purify the fusion proteins of glutathione S-transferase (GST)-N-terminal of histone deacetylase4 (HDAC4-N') (1-1952 bp) and GST- C-terminal of HDAC4 (HDAC4-C') (1708-3255 bp) in E.coli.

METHODSThe DNA fragments (HDAC4-N' and HDAC4-C') amplified by PCR were ligated into GST fusion vector (pGEX-6P-1) to construct the recombinant plasmids. After identification with restriction digestion and DNA sequencing, the recombinant plasmids were transformed into E.coli BL21 and induced by IPTG for their expression. After identification by SDS-PAGE and Western blotting, the target proteins were purified by glutathione sepharose 4B.

RESULTSThe results of restriction digestion and DNA sequencing confirmed successful construction of the recombinant plasmids. The relative molecular masses of the fusion proteins were approximately 110500 and 93080 as shown by SDS-PAGE. Western blotting demonstrated that the fusion proteins could be recognized by the specific anti-HDAC4 antibody.

CONCLUSIONWe have successfully constructed the recombinant expression vectors of pGEX-6P-1/HDAC4-N' and pGEX-6P-1/HDAC4-C' and induced the expression of the fusion proteins, which may facilitate functional studies of HDAC4 with other proteins.

Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Histone Deacetylases ; biosynthesis ; genetics ; Humans ; Peptide Fragments ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Repressor Proteins ; biosynthesis ; genetics

Prokaryotic expression vector of mouse HPV16E6 gene was constructed. A pair of primers were designed according to the digestion sites in plasmid pGEX-KG and the HPV16E6 gene sequence published by GenBank. The DNA fragment of 321bp was amplified by PCR from the HPV recombinant plasmid with HPV16E6 gene, then cloned into pGEX-KG and transformed into the host E. coli strain JM109. The fragment was conformed to the original sequence, which indicated that fusion expression vector pGEX-KG-HPV16E6 was constructed. The pGEX-KG-HPV16E6 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degrees C, the expression product of HPV16E6 gene was identified by SDS-PAGE and Western blot. HPV16E6 fusion protein had been expressed successfully in the form of inclusion bodies, the molecular weight of fusion protein being 38 kD. Meanwhile, the optimum condition of HPV16E6 fusion protein expression was induced with 1.0 mmol/L IPTG for 4h. The fusion protein reacted specifically with the antibodies against HPV16E6. HPV16E6 gene was successfully expressed in E. coli, which could be used as a basis for preparing HPV16E6 vaccine in human.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

Bcel-2 family proteins (Bcl-x(L), Bcl-2, Mel-1 etc.) are key regulators of some life processes, including apoptosis and autophagy. They are currently considered as promising targets for developing new anti-tumor therapies. In our study, the human Bcl-2/Bcl-x(L) chimeric gene and the human/mouse Mel-1 chimeric gene were designed and cloned, and the prokaryotic expression vectors for expressing glutathione S-transferase (GST) fusion proteins and histidine tag fusion proteins were constructed respectively. These two proteins as well as the GST-Bcl-x(L) fusion protein were all successfully expressed in E. coli and subsequently purified. In addition, we measured the binding of these Bcl-2 family proteins to the Bid BH3 peptide by fluorescence polarization-based assay. The dissociation constants (Kd) obtained by us were in general agreement with the data reported in literature. The Kd values of all three proteins with or without the GST tag were almost identical. All these results validate the biological functions of these Bcl-2 family proteins obtained by us. These proteins can be used in the experimental screening of small-molecule regulators of Bcl-2 family proteins in vitro.
Escherichia coli ; genetics ; metabolism ; Fluorescence Polarization ; methods ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; bcl-X Protein ; biosynthesis ; genetics ; isolation & purification

The aim of the study is to obtain an efficient expression of recombinant ubiquitin-like specific protease 1 (Ulp1) by gene engineering. We cloned the Ulp1p, active fragment (403 aa-621 aa) of Ulp1, from Saccharomyces cerevisia, and subcloned into pGEX/Rosetta (DE3) to form an expression plasmid, pGEX-Ulp1p-His6. In order to enhance the solubility of GST-Ulp1p-His6, we purified the fusion protein GST-Ulp1p-His6 by either glutathione S-transferase agarose or Ni-NTA resin chromatography, the purity was up to 98%. We utilized the protein to cleave the SUMO fusions, and the specific activity of GST-Ulp1p-His6 was 1.375 x 10(4) U/mg. This study showed that the recombinant protein GST-Ulp1p-His6 displayed high specificity and activity.
Cloning, Molecular ; Cysteine Endopeptidases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; biosynthesis ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; enzymology ; Solubility

PHD finger protein 8 (PHF8) is a novel protein with PHD domain and Jmjc domain, which may play important role in regulating transcription and histone demethylation. It is necessary to generate the antibody against PHF8 in order to further study its biological function. First we constructed plasmid pET41b-PHF8 (aa886-936) and expressed the GST-PHF8 (aa886-936) fusion protein in Escherichia coli BL21. We then purified the fusion protein by Glutathione Sepharose 4B beads and subjected to immunize the rabbits for acquiring antiserum. We obtained PHF8 polyclonal antibody by affinity purifying the antiserum with CNBr-activated Sepharose 4B beads. The antibody was effective in Western blotting and immunofluorescence with high specificity. Immunofluorescence also showed that PHF8 protein was located in nucleus in HeLa cells.
Animals ; Antibodies ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; immunology ; Histone Demethylases ; biosynthesis ; genetics ; immunology ; Immunization ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transcription Factors ; biosynthesis ; genetics ; immunology

In the present study, we constructed a prokaryotic expression vector containing SOX4 protein encoding sequences. The GST-SOX4 soluble protein was expressed in Escherichia coli DH5alpha and purified by glutathione sepharose-4B. The purified recombinant protein was used to immunize Balb/C mice and the monoclonal antibody against SOX4 was prepared by using hybridoma technique. The titer of the antibody was determined as 1 x 10(5) by indirect ELISA. The specificity of the antibody was verified by Western blotting analysis. The monoclonal antibody specifically recognized the overexpressed exogenous SOX4 protein as well as endogenous SOX4 protein. The expression level of SOX4 protein in different cell lines and mouse tissues was detected by using the antibody. Differential expression of the protein was demonstrated by Western blotting. The data indicated that the antibody was specific. The antibody can be used as an important tool for further exploration of the role of SOX4 in tumorigenesis.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Glutathione Transferase ; genetics ; metabolism ; Humans ; Isoenzymes ; genetics ; metabolism ; Liver Neoplasms ; metabolism ; Lung Neoplasms ; metabolism ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; SOXC Transcription Factors ; genetics ; immunology ; metabolism ; Tumor Cells, Cultured