[Isolation and characterization of an alkaline xylanasefrom a newly isolated Bacillus sp. QH14].

Twenty-fivealkaline xylanase producing strains were isolated from Qinghai Lake side soil samples. Among these strains, QH14 produced 648.79 U/mLxylanase, and the enzymatic specific activity was 1148.56 U/mg after purification. This alkaline xylanase producing strain belongs to genus Bacillus based on16S rDNA sequencing analysis and then was designated as Bacillus sp. QH14. The alkalinexylanaseencoding gene, XynQH14, was cloned from Bacillus sp. QH14 and expressed in Escherichiacoli BL21 (DE3). The specific activity of the recombinant xylanase XynQH14 was 700.47 Umg-1 after purification by Ni-NTA affinity chromatography. The optimal temperature and pH of XynQH14 were 60℃ and pH9.2, respectively. Its activity was 50% of initial activity after incubation at 55 ℃ for 1h, 80% at pH7-11 at 37 ℃ for 24 h, and 31.02% at pH11 at 50℃ after 24 h, indicating that XynQH14 isthermostable and alkali-stable. These properties ofXynQH14 suggest its favorable potential applications in pulp and paper industries.

Fusion of HSA influences TNF-α neutralizing activity of shTNFRs.

Soluble human tumor necrosis factor receptors (shTNFRI and shTNFRII) are antagonists of tumor necrosis factor-α (TNF-α) and are under clinical investigation as therapy for autoimmune diseases and transplant rejection. However, shTNFRI and shTNFRII are limited for clinical usage because of their short half-lives in vivo. Recombinant TNF-α receptors (infliximab and etanercept) are used in treatment of rheumatoid arthritis and Crohn's disease but are also being tested for a number of other autoimmune diseases. Human serum albumin (HSA) has been used to construct long-acting fusion proteins. Here, we report the effect of fusion of HSA with shTNFRI and with shTNFRII on shTNFR's neutralizing activity against TNF-α. HSA fusion proteins were separately expressed in Pichia pastoris. Purified recombinant shTNFRI-HSA, HSA-shTNFRI and HSA-shTNFRII could block the cytolytic activity of TNF-α in L929 cells, and the fusion at N-terminus of shTNFRI could result in larger degree of activity decline than that at the C-terminus. Activity of three fusion proteins was much weaker than etanercept, which demonstrated that fusion of HSA significantly influenced TNF-α neutralizing activity of shTNFRs. Compared with Fc fragment, HSA fusion technology may therefore not be an ideal strategy in development of long-acting shTNFRs protein drugs.

MAFIP is a tumor suppressor in cervical cancer that inhibits activation of the nuclear factor-kappa B pathway.

Cervical cancer is the second most common cancer in women. Inactivation of tumor suppressor genes underlies the transformation and progression of cervical cancer. Previously, we reported MAFIP can inhibit the growth of human cervical cancer HeLa cells. In this study, MAFIP was found to be downregulated in cervical intraepithelial neoplasia tissues. Induced expression of MAFIP in HeLa cells strongly inhibited tumor formation in nude mice, confirming its tumor suppressor activity in vivo. Overexpression of MAFIP inhibited activation of the NF-κB pathway, a commonly active pathway in cancer cells, by preventing the phosphorylation of IKK and IκBα, degradation of IκBα and the nuclear localization of p65. Induction of c-myc, an oncogene controlled by NF-κB, was severely impaired in the cells overexpressing MAFIP. In contrast, knockdown of MAFIP by siRNA activated the NF-κB pathway and promoted cell proliferation. These data suggest MAFIP functions as a tumor suppressor in cervical cancer in part by inhibiting activation of the NF-κB pathway.

High level expression of a truncated ß-mannanase from alkaliphilic Bacillus sp. N16-5 in Kluyveromyces cicerisporus.

A truncated alkaline ß-mannanase from alkaliphilic Bacillus sp. N16-5 (MAN330) was expressed and secreted in Kluyveromyces cicerisporus. The recombinant engineered strain for MAN330 production was stable during 80 generations, and the maximum yield of MAN330 reached 3,795 U/ml in 15 l fermenter. MAN330 exhibited similar pH optima, temperature optima, and substrate specificities to its full-length protein (MAN493). However, stability of MAN330 was about 7% higher than that of MAN493 from pH 9-11. MAN330 had about 10% higher stability than MAN493 from 60°C to 80°C.

Mycobacterium tuberculosis antigen Wag31 induces expression of C-chemokine XCL2 in macrophages.

Tuberculosis is still a major threat to human health. To date, only approximately half of the proteins encoded by Mycobacterium tuberculosis H37Rv have been assigned specific functions. Wag31 (Rv2145c) is one of the bacterial proteins whose function is mostly unknown. Using a modified split-ubiquitin membrane yeast two-hybrid system, we screened a macrophage cDNA library with Wag31 as bait and identified XCL2, a C-subfamily chemokine, as a binding partner for Wag31. More importantly, Wag31 was found to specifically stimulate XCL2 expression in macrophages. The results from this study demonstrate that expression of C-chemokine is not restricted to certain types of T cells and natural killer cells. Because C-chemokine is chemotactic for CD8+ and CD4+ T cells, our novel findings could provide a new mechanism by which the bacteria induce cell-mediated immunity and by which Wag31 could be a potential target for controlling M. tuberculosis infection.

Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic effects of BNIP3 in two human cell lines.

BNIP3 is a unique pro-apoptotic protein which belongs to the BH3-only subset of the Bcl-2 family and localizes on mitochondrial membrane. Despite the inherent difficulty of identifying binding partners for membrane proteins, several binding partners for BNIP3 have been identified. In this study, a modified split-ubiquitin membrane yeast two-hybrid system was constructed and used to identify acetyl-Coenzyme A acyltransferase 2 (ACAA2) as a new BNIP3 binding partner. The interaction between BNIP3 and ACAA2 was confirmed by pull-down and co-immunoprecipitation assays. ACAA2 was also found to co-localize with BNIP3 in mitochondria. Furthermore, the apoptosis induced by over-expressed BNIP3 via transfection or hypoxia treatment was abolished by ACAA2 in human hepatocellular carcinoma HepG2 cells and osteosarcoma U-2 OS cells. These results strongly suggest that ACAA2 be a functional BNIP3 binding partner and provide a possible linkage between fatty acid metabolism and apoptosis of cells.

The orphan nuclear receptor Rev-erbbeta recruits Tip60 and HDAC1 to regulate apolipoprotein CIII promoter.

Nuclear hormone receptors function as ligand activated transcription factors. Ligand binding and modification such as acetylation have been reported to regulate nuclear hormone receptors. The orphan receptors, Rev-erbalpha and Rev-erbbeta, are members of the nuclear receptor superfamily and act as transcriptional repressors. In this study, the role of recruitment of co-factors by Rev-erbbeta and acetylation of Rev-erbbeta in modulating apolipoprotein CIII (apoCIII) transcription were investigated. Rev-erbbeta was found to transcriptionally repress apoCIII after binding to the apoCIII promoter. Tip60, a histone acetyl-transferase (HAT), was a novel binding partner for Rev-erbbeta and recruited to the apoCIII promoter by Rev-erbbeta. Tip60 was able to acetylate Rev-erbbeta and relieve the apoCIII repression mediated by Rev-erbbeta. This de-repression effect depended on acetylation of Rev-erbbeta at its RXKK motif by Tip60. In addition, histone deacetylase 1 (HDAC1) interacted with Rev-erbbeta and was recruited to the apoCIII promoter by Rev-erbbeta to antagonize Tip60's activity. Taken together, we have provided evidence that Rev-erbbeta modulates the apoCIII gene expression by recruiting different transcription co-activator or co-repressor.

A zinc finger HIT domain-containing protein, ZNHIT-1, interacts with orphan nuclear hormone receptor Rev-erbbeta and removes Rev-erbbeta-induced inhibition of apoCIII transcription.

The orphan receptors, Rev-erbalpha and Rev-erbbeta, are members of the nuclear receptor superfamily and specifically repress apolipoprotein CIII (apoCIII) gene expression in rats and humans. Moreover, Rev-erbalpha null mutant mice have elevated very low density lipoprotein triacylglycerol and apoCIII levels. However, ligands for Rev-erb are unknown and the regulatory mechanism of Rev-erb is poorly understood. Conceivably, cofactors for Rev-erb may play an important role in the regulation of lipid metabolism. In this study, a zinc finger HIT domain-containing protein, ZNHIT-1, interacted with Rev-erbbeta. ZNHIT-1 was found to be a conserved protein in eukaryotes and was highly abundant in human liver. Furthermore, ZNHIT-1 was identified as a nuclear protein. Serial truncated fragments and substitution mutations established a putative nuclear localization signal at amino acids 38-47 of ZNHIT-1. A putative ligand-binding domain of Rev-erbbeta and the FxxLL motif of ZNHIT-1 were required for their interaction. Finally, ZNHIT-1 was recruited by Rev-erbbeta to the apoCIII promoter and removed the Rev-erbbeta-induced inhibition of apoCIII transcription. These findings demonstrate that ZNHIT-1 functions as a cofactor to regulate the activity of Rev-erbbeta, and may play a role in lipid metabolism.

Cloning, expression and characterization of human tissue-specific DNA polymerase lambda2.

DNA polymerase (POL) lambda plays an important role during DNA repair and DNA nonhomologous recombination processes. A novel POL lambda variant was cloned from a human liver cDNA library and named POL lambda2 (GenBank Accession No. AY302442). POL lambda2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL lambda2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL lambda2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL lambda and POL lambda2 in normal liver tissues and hepatocellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL lambda. This abnormality might be involved in hepatocarcinogenesis. The recombinant POL lambda2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chromatography in an FPLC system. The analysis using isotope a-(32)P-dCTP incorporation in vitro showed that the purified recombinant POL lambda2 exhibited DNA polymerase activity.

Characterization of human dopamine responsive protein DRG-1 that binds to p75NTR-associated cell death executor NADE.

Expression of human dopamine responsive gene-1 (DRG-1) is up-regulated in response to treatment of dopamine in the rat astrocytes. However, its functions are not clear up to now. In the presented studies, DRG-1 was identified to be a conserved gene in the vertebrate and expressed abundantly in human testis, brain and skeletal muscle. DRG-1 was shown to interact with human p75NTR-associated cell death executor (NADE) in vivo and in vitro, and the interaction occurred in cytoplasm. The regions required for the interaction were subsequently mapped to the N-terminal of DRG-1 and the C-terminal of NADE. Furthermore, MTT assay showed that stable expression of DRG-1 in 293 cells could promote cell proliferation, and this promotion was suppressed by overexpression of NADE. In flow cytometry cell cycle analysis, overexpression of DRG-1 in 293 or PC12 cells increased the population of cells in the S phase with a concomitant decrease in G0/G1 population. These findings suggest that DRG-1 may contribute to the dopamine-induced cell growth, which is negatively regulated by NADE.

Human homologue of SETA binding protein 1 interacts with cathepsin B and participates in TNF-Induced apoptosis in ovarian cancer cells.

Lysosomal cysteine protease cathepsin B has been reported to play an important role in apoptosis of many different cancer cells, but the regulation of cathepsin B in apoptosis is poorly understood. Human homologue of SETA binding protein 1 (hSB1) was identified to interact with cathepsin B by yeast-two hybrid method, and the interaction was confirmed in vitro GST pull-down assay and in vivo coimmunoprecipitation experiment. hSB1 was co-localized with cathepsin B in cellular lysosomes. Our previous study has shown that TNF can induce ovarian cancer cells OV-90 apoptosis and the apoptosis process is cathepsin B-depended. Here we provide evidence that overexpression of cathepsin B-interacting protein hSB1 could suppress TNF-triggered apoptosis in OV-90 cells, but has no effect on cellular cathepsin B activity. hSB1 may function as a regulator of cathepsin B-mediated apoptosis.

The novel human gene MIP functions as a co-activator of hMafF.

The human transcription factor MafF (hMafF) lacks a transactivation domain, it contains a heptad-leucine repeat motif (viz., Leu-zipper) that may mediate protein-protein interactions to regulate transcriptional activities. A protein with a coiled-coil domain encoded by a novel human gene (GenBank Accession No. AF289559) was found to interact with hMafF in vitro and in vivo and is designated as a MafF interacting protein (MIP). Here, we provide evidence that the coiled-coil domain of MIP is essential for binding to the Leu-zipper of hMafF and that the interaction between MIP and hMafF causes the translocalization of MIP from cytoplasm to nucleolus in HELA cells. We used a promoter-reporter system containing six tandem repeats of the US2 element, located in the promoter of the human oxytocin receptor gene and reported to bind specifically to hMafF, to understand effects on transcriptional activation of hMafF, and its interaction with MIP. Expression of hMafF or MIP alone did not alter basal reporter transcription activity, whereas co-expression of hMafF and MIP activated transcription efficiently. Moreover, truncated MIP, still containing the coiled-coil domain, transactivated as well as the full-length MIP did, and highlighting that MIP acts as a co-activator of hMafF.

Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae.

Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R' was co-transformed with a PCR amplified expression cassette of human IFNalpha2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNalpha2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNalpha2b, the expression plasmid pHC11R-IFNalpha2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R-IFNalpha2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.

Construction, expression and characterization of human interferon alpha2b-(G4S)n-thymosin alpha1 fusion proteins in Pichia pastoris.

AIM: Interferon alpha2b (IFNalpha2b) and thymosin alpha1 (Talpha1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNalpha2b and Talpha1 linked by different lengths of (G4S)n (n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex 75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNalpha2b monoclonal antibody and Talpha1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNalpha2b and immunomodulatory activity of Talpha1 in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.

Expression of human plasminogen kringle 5 as fusion protein with truncated hIFNgamma gene in Escherichia coli.

The human interferon gamma (hIFNgamma) gene was used as a fusion partner to mediate the expression of heterologous proteins and the effect of the fusion partner length on the expression of the heterologous protein was researched. Plasminogen kringle 5 (pk5), an inhibitor of angiogenesis, was fused to hIFNgamma and its serially truncated fragments, respectively, and the expression of fusion proteins was determined by SDS-Page gel. The pk5 protein was obtained readily by the introduction of sequences recognized by protease factor Xa at the fusion site and ion-exchange chromatography was employed to purify pk5. The recovery of the biological activities of pk5 was studied using the orthogonal experimental design L9 (3(4)) (four factors, three levels, nine experiments) and evaluated by measurement of anti-endothelial cell proliferation in vitro.

Purification and Characterization of Recombinant Hepatitis B Virus Surface Antigen SS1 Expressed in Pichia pastoris.

The purification of recombinant hepatitis B surface antigen, SS1 protein, expressed in Pichia pastoris, and the investigation of its physiochemical characters and immunogenicity were described here. Employing McAb immunoaffinity chromatography, this protein was purified to purity of 95%. The results of ELISA and Western blotting showed good antigenicity of this purified SS1 protein. CsCl gradient centrifugation and electron microscopy assay proved that this purified protein could be assembled into particles similar to the HBV subviral particles. Strong antibody responses against both the HBs and PreS1 epitopes were induced in BALB/c mice immunized with this purified protein. The simultaneous injection of a CpG adjuvant induced a Th1-like immune response against both the HBs and PreS1 epitopes.

Expression of the Recombinant Hepatitis B Virus Surface Antigen Carrying PreS Epitopes in Pichia pastoris.

Many studies have suggested that hepatitis B surface antigen(HBsAg) including PreS sequences could be an ideal candidate for highly effective hepatitis B virus vaccine. Modified surface antigens S1S, SS1 and S2S which carried PreS epitopes, were expressed in Pichia pastoris. The characterization of antigenicity and particle assembly demonstrated that the expression products could be assembled into particles which presented S, PreS1 or PreS2 antigenicity, respectively. The expression was more efficient than that in Saccharomyces cerevisiae.