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

Animals ; Glutathione Transferase ; biosynthesis ; Liver Neoplasms, Experimental ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo detect expression of p-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and glutathione S-transferase (GST-pi), and to evaluate its clinical significance in neuroblastoma (NB).

METHODSSP immunohistochemical technique was used to investigate expression of P-gp, MRP, and GST-pi in 70 cases of NB.

RESULTSThe frequency of expression of P-gp, MRP, and GST-pi was 61.4%, 38.6%, and 51.4%, respectively. The coexpression rate of P-gp and MRP, P-gp and GST-pi, MRP and GST-pi, P-gp, MRP and GST-pi was 32.9%, 35.7%, 27.1%, and 24.3%, respectively. Significant positive correlation was observed between P-gp and MRP expression (P = 0.001), and between MRP and GST-pi expression (P = 0.012), but no correlation was found between P-gp and GST-pi expression. The expression of P-gp and MRP was higher in tumors from patients over 1 year old compared with those less than 1 year old at diagnosis (P = 0.01, 0.018, respectively). MRP expression was higher in tumors from the metastatic than the non metastatic groups (P = 0.015). All tested proteins showed significant relationship to the differentiation of the tumor (P = 0.006, 0.000, 0.019, respectively), but no correlation was found to the stage of NB or sex of the patients. MRP expression was significantly related to the reduction of both median survival time and the two-year cumulative survival (P = 0.02). In contrast, P-gp and GST-pi expression had no correlation with survival.

CONCLUSIONSThe intrinsic multidrug resistance of NB involves the combined effects of P-gp, MRP, and GST-pi. MRP expression may be an important parameter in predicting the prognosis of patients with NB.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; Adolescent ; Child ; Child, Preschool ; Female ; Gene Expression ; Glutathione S-Transferase pi ; Glutathione Transferase ; biosynthesis ; Humans ; Immunohistochemistry ; Infant ; Isoenzymes ; biosynthesis ; Male ; Multidrug Resistance-Associated Proteins ; biosynthesis ; Neuroblastoma ; metabolism ; mortality ; Survival Rate

Recombinant proteins were expressed as fusions with the phagemid system of pHEN-KM13 and the characteristics and activities of the fusion proteins displayed on the surface of filamentous phagintain the were studiedon abilty. The altered titer of rescued phages from the phagemid system after trypsin treatment indicated the relative quantity of the phages displaying fusion proteins. The rescue phages displayed foreign proteins could keep the bacterial infection ability, while the bald phage without foreign protein displayed on its surface was sensitive to trypsin treatment and lost the bacterial infection ability. To determine the upper limit for filamentous phage display, four recombinant proteins, glutathione-S-transferase and glutathione-S-transferase fused with three various size peptide linkers, were fused to N terminus of capsid protein gp3 and rescued by helper phage KM13. The rescued phages which displayed fused protein with the size of 40kD or less maintain the infection ability. To assay the activity of the phage displayed protein, the known small molecule probe was used in the interaction study with protein incorporated on phage surface. Results showed that the glutathione-S-transferase on phage surface still bound to glutathione specifically. It indicated that the glutathione-S-transferase displayed on phage surface was correctly folded and functionally active. The results demonstrated that it was feasible to use small molecule probes to interact with the protein displayed on phage surface. In turn, the method described here also demonstrated that phage display system could be utilized to investigate the interactions between protein and small molecules.
Glutathione ; genetics ; metabolism ; Glutathione Transferase ; biosynthesis ; genetics ; Helper Viruses ; genetics ; metabolism ; Inovirus ; genetics ; metabolism ; Peptide Library ; Protein Binding ; Protein Interaction Domains and Motifs ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

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

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; growth & development ; Glutathione Transferase ; biosynthesis ; genetics ; Heparin Lyase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo explore the correlation of chemotherapy efficacy in tongue squamous cell carcinoma(SCC) with expression level of P-glycoprotein(Pgp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathiones-tranferase (GST-pi), DNA topo-isomerase II alpha (Topo II alpha).

METHODSThe expression patterns of Pgp, MRP, LRP, GST-pi and Topo II alpha in 40 patients (pre and post-chemotherapy, respectively) with tongue SCC were examined by immunohistochemically labelled streptavidin bioein method (LsAB).

RESULTSThe expression ratios of Pgp, MRP, LRP, GST-pi and Topo II alpha in pre-chemotherapy cases were 47.5%, 50%, 35%, 45%, 82.5%, respectively. No relations between expression of Pgp, MRP, LRP, GST-pi, Topo II alpha and clinic indexes were established (P > 0.05). Expression ratios of Pgp, MRP in post-chemotherapy cases were higher than that in pre-chemotherapy cases (P < 0.05). Expression of Pgp and MRP showed relevance with drug resistance (P < 0.05). The co-expression was common, the ratios of co-expression of Pgp, MRP, GST-pi and MRP, GST-pi in chemotherapy non-responders were 40% and 50%, respectively, but 0 in responders.

CONCLUSIONThe intrinsic multidrug resistance of tongue SCC is relevant to the effects of Pgp, MRP, GST-pi.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Adult ; Antigens, Neoplasm ; Carcinoma, Squamous Cell ; metabolism ; DNA Topoisomerases, Type II ; biosynthesis ; genetics ; DNA-Binding Proteins ; Female ; Glutathione S-Transferase pi ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Isoenzymes ; biosynthesis ; genetics ; Male ; Middle Aged ; Multidrug Resistance-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Random Allocation ; Tongue Neoplasms ; metabolism ; Vault Ribonucleoprotein Particles ; biosynthesis ; genetics

OBJECTIVETo investigate the reversal effect of haloperidol (Hal) on doxorubicin (Dox) resistance and its inhibition effect on P-glycoprotein and swelling-activated chloride channel in Dox-resistant erythro-leukemic cell line K562/Dox.

METHODSTumor cell proliferation was measured by LDH assay. mRNA expressions of P-glycoprotein (MDR1), glutathione S-transferase Pi (GSTpi) and MDR-associated protein (MRP) of K562/Dox treated with Hal were assayed by RT-PCR. Chloride-sensitive dye MQAE was loaded into K562/Dox cells and the intracellular fluorescence intensity was measured to evaluate the effect of Hal on chloride channel in swelling-activated K562/Dox cells. Coulter counter ZM and Channelyzer 256 were used to measure cell volume regulation.

RESULTSHal significantly reversed Dox resistance in K562/Dox cells after 12.50, 6.25 and 3.12 micromol/L Hal treatment, the chemosensitivity to Dox increased by 8.61, 4.35 and 2.25 times respectively. After treatment with Hal 12.50 micromol/L, MDR1 and MRP mRNA expression were gradually down-regulated in a time-dependent manner on d1-d3, reducing to 76.3% and 64.6% of the control level on d3 (P < 0.05), while GSTpi mRNA expression decreased by 66.1% (P < 0.05) on d1-d2, and began to recover on d3. The swelling-activated chloride channel and cell regulatory volume decreased (RVD) in K562/Dox cells were also inhibited by Hal. Under hypotonic challenge the cellular fluorescence intensity which represented chloride concentration declined by (34.46 +/- 5.91)%. After adding 6.25 micromol/L and 18.75 micromol/L Hal, the hypotonic challenge only caused decrease in fluorescence intensity by (24.43 +/- 3.25)% and (16.63 +/- 4.98)% (P < 0.01). RVD in hypotonic condition was (84.95 +/- 5.69)%. RVD under hypotonic condition with 6.25 micromol/L and 18.75 micromol/L Hal were (51.12 +/- 6.01)% and (39.51 +/- 4.79)% respectively (P < 0.01).

CONCLUSIONA nontoxic concentration of haloperidol can significantly reverse drug resistance through a multi-pathway effect, including down-regulating mRNA expressions of MDR, GSTpi and MRP, inhibition of swelling-activated chloride channel and RVD in K562/Dox cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Antibiotics, Antineoplastic ; pharmacology ; Chloride Channels ; drug effects ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Glutathione S-Transferase pi ; Glutathione Transferase ; biosynthesis ; genetics ; Haloperidol ; pharmacology ; Humans ; Isoenzymes ; biosynthesis ; genetics ; K562 Cells ; Multidrug Resistance-Associated Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

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

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