Tissue factor pathway inhibitor (TFPI) is one of the major physiological inhibitors of the human blood coagulation cascade and may have great potential in the prevention and therapy of diseases caused by thrombus formation. In this study, recombinant human tissue factor was generated in E. coli containing a recombinant vector constructed by inserting TFPI cDNA into pGEX-2T vector. The generated recombinant TFPI (rTFPI) could be simply purified with glutathione-agarose affinity method and maintained its biological function in terms of inhibition of tissue factor and factor Xa.
Escherichia coli ; genetics ; metabolism ; Humans ; Lipoproteins ; biosynthesis ; genetics ; pharmacology ; Peptide Fragments ; biosynthesis ; genetics ; pharmacology ; Plasmids ; Recombinant Proteins ; biosynthesis ; pharmacology ; Transfection

In this study, peaT1 gene was subcloned into the Pichia pastoris expression vector pPIC9K, which contained both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pPIC9K-peaT1. The recombinant plasmid was linearized by Sal I or Bgl II and transformed into P. pastoris GS115 by electroporation method. Recombinant strain was screened by Minimal Dextrose Medium and further confirmed by PCR. The gene was in frame integrated into the Pichia genome through homologous recombination, resulting the recombinant strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant protein was expressed and secreted into the supernatant. The SDS-PAGE analysis indicated that the apparent molecular weight of target protein was about 35 kD. Bioassay results showed that the inhibition rate of the expressed protein against TMV was 30.37%. The supernatant was collected and then purified by anion exchange chromatography. This protein can promote seedling growth of wheat obviously.
Alternaria ; genetics ; Fungal Proteins ; biosynthesis ; genetics ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Triticum ; drug effects ; growth & development

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.
Bacterial Proteins ; biosynthesis ; genetics ; Fermentation ; Fungal Proteins ; biosynthesis ; genetics ; Lipase ; biosynthesis ; genetics ; Oxygen ; analysis ; pharmacology ; Pichia ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; Truncated Hemoglobins ; biosynthesis ; genetics

The total RNA was extracted from peripheral blood mononuclear cells (PBMC) which was isolated from Meishan porcine and induced with concanavaline A (ConA), then the porcine interferon gamma gene (PoIFNgamma, 501bp) was amplified by RT-PCR. The result of sequencing demonstrated that the amplified PoIFNgamma had 100% nucleotide homology with the other porcine IFNgamma sequence published on GenBank. The objective gene (PoIFNgamma) was inserted into adenoviral shuttle vector, pShuttle-CMV, to construct recombinant plasmid pSh-PoIFNgamma. And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37 degrees C for 24h on kanamycin resistance plate and selected for smaller colonies. Then, the extracted recombinant plasmid was named pAd-Sh-PoIFNgamma, which was confirmed by Pac I digestion, and transformed into XL10-Glod(r) for copious preparation. pAd-Sh-PoIFNgamma linearized with Pac I was co-transfected with liposome into 293 package cell-line. After 7d-10d, the typical cytopathic effect indicated that recombinant adenoviral genome (deleted with E1 and E3 genes) carrying PoIFNgamma was successfully packaged into intact virion. The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR. The antiviral activity of PoIFNgamma was tested by CPE50 method. The results showed that the PoIFNgamma expressed by adenovirus had high antiviral activity, which was 1.3 x 10(6) U/mL against VSV in MDBK cells. The results demonstrated that the recombinant adenovirus carrying PoIFNgamma could be stably passaged.
Adenoviridae ; genetics ; Animals ; Antiviral Agents ; pharmacology ; Interferon-gamma ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; pharmacology ; Swine

In order to express PebC1 in Pichia pastoris, the pebC1 sequence was amplified from genome Botrytis cinerea BC-4-2-2-1 by PCR and subcloned into the Pichua pastoris expression vector pPIC9K to generate pPIC9K-pebC1. The recombinant plasmid was linearized by Bgl II and transformed into Pichia pastoris GS115 by electroporation. Recombinant Pichia pastoris GS115/pPIC9K-pebC1 was screened by MD and G418-YPD plates and further confirmed by PCR. The protein expression was induced by methanol and analyzed by SDS-PAGE. SDS-PAGE analysis showed a special band about 39 kDa and western blotting indicated a good antigenicity of the expressed protein. Bioassay results showed that the recombinant protein PebC1 can induce resistance to gray mould disease of cucumber and Arabidopsi thaliana.
Botrytis ; chemistry ; Fungal Proteins ; biosynthesis ; genetics ; pharmacology ; Pichia ; genetics ; metabolism ; Plant Diseases ; prevention & control ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.
Antioxidants ; pharmacology ; DNA, Complementary ; Electroporation ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Serpins ; biosynthesis ; genetics

In order to determine the adjuvant effects of the chicken IL-2 (ChIL-2) on new generation vaccines, ChIL-2 gene was amplified from ConA-stimulated chicken spleen cells by RT-PCR and was directionally inserted into fowlpox virus (FPV) transferring vector p1175 under the control of FPV early/late promoter (PE/L), resulting in recombinant transferring vector p1175IL2. Then the p1175IL2 plasmid was transfected into chicken embryo fibroblasts (CEF) pre-infected with wild type FPV to generate recombinant fowlpox virus expressing ChIL-2 (rFPV-IL2). By selection of blue plaques on the CEF, overlaid with agar containing X-gal, rFPV-IL2 was obtained and purified. The supernatant from CEF monolayer infected with rFPV-IL2 (M.O.I2.0) after 72 hours was detected for the production of ChIL-2 by XTT/PMS colorimetric assay. About 3.6 x 10(5) u/mL of specific ChIL-2 activity was determined. The results show that rFPV-IL2 can express ChIL-2 effectively. rFPV-IL2 provides us with an effective tool for studying avian immunology as well as a potential vaccine-enhancing agent.
Animals ; Chick Embryo ; Chickens ; Fowlpox virus ; genetics ; Interleukin-2 ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; pharmacology

Six 89bp primers were designed on the base of the cDNA sequence encoding the human leptin reported on the NCBI. The synthetic gene with 464bp encoding rhLep was obtained by SOE ( splicing by overlap extension) PCR. The expression vector pET22b(+)/rhLep was constructed and transformed into E. coli BL21 (DE3). The rhLep protein was expressed as inclusion bodies with the yield of more than 50% of total bacterial proteins after IPTG induction. The rhLep protein, which has a molecular weight about 16kD, was purified by Ni2+ affinity chromatography column and identified by SDS-PAGE. The MTT Assay shows that rhLep promotes EC304 cells growth at the low concentration of 10ng/mL to 30 ng/mL, and rhLep appears cytotoxic to EC304 cells with the high dose of 50ng/mL to 225ng/mL. The viability of EC304 cells decreases to 1.2% with the concentration of 225ng/mL of rhLep. The massive apoptosis of rhLep on EC304 cells is observed by AO-staining under fluorescent microscope. All these results would lay the foundation for the further study of its biological functions in vitro and in vivo.
Apoptosis ; drug effects ; Escherichia coli ; genetics ; Humans ; Leptin ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

Porcine interferon-alpha (pIFN-alpha) fermentative production by recombinant Pichia pastoris was carried out in a 10-L bioreactor to study its metabolism changes and effects on fermentation under different inducing strategies, by analyzing the change patterns of the corresponding metabolism and energy regeneration. The results show that the specific activities of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) largely increased when reducing temperature from 30 degrees C to 20 degrees C under pure methanol induction, leading significant enhancements in methanol metabolism, formaldehyde dissimilatory energy metabolism and pIFN-alpha antiviral activity. The highest pIFN-alpha antiviral activity reached 1.4 x 10(6) IU/mL, which was about 10-folds of that obtained under 30 degrees C induction. Using methanol/sorbitol co-feeding strategy at 30 degrees C, the major energy metabolism energizing pIFN-alpha synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle, formaldehyde dissimilatory pathway was weakened and accumulation of toxic intermediate metabolite-formaldehyde was relieved, and methanol flux distribution towards to pIFN-alpha synthesis was enhanced. Under this condition, the highest pIFN-alpha antiviral activity reached 1.8 x 10(7) IU/mL which was about 100-folds of that obtained under pure methanol induction at 30 degrees C. More important, enhanced pIFN-alpha production with methanol/sorbitol co-feeding strategy could be implemented under mild conditions, which greatly reduced the fermentation costs and improved the entire fermentation performance.
Animals ; Energy Metabolism ; Fermentation ; Interferon-alpha ; biosynthesis ; genetics ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Sorbitol ; pharmacology ; Swine

The thrombolytic agent DSPAalpha1 is currently undergoing clinical trials for the treatment of acute ischemic stroke and has shown good pharmacodynamic, pharmacokinetic and safety profiles. Here, the DSPAalpha1 gene, optimized for the preferred codons of yeast, was cloned into the Pichia pastoris strains GS115 and KM71. Both expression systems produced functional DSPAalpha1 into the broth medium under shaking flask growth conditions with the yield of about 70 mg/L, and 105 mg/L, respectively. In addition, three glycosylation minus DSPAalpha1 mutants, constructed by site-directed mutagenesis, were also expressed in Pichia pastoris. The mutant proteins were assayed by SDS-PAGE and fibrin degradation activities were evaluated. The secretion levels of all the mutants, especially N362Q and N117Q/N362Q, were so lower compared to the wild-type DSPAalpha1 that only minimal quantities of mutant protein could be recovered by purification from the culture medium. The protein specific activities from mutants (N117Q, N362Q) were less 25% than that of the wild type protein. These results imply that the N-linked carbohydrate chains (at N117 and N362) are vital for the enzymatic activity of rDSPAalpha1 and for its secretion from Pichia pastoris.
Fibrinolytic Agents ; metabolism ; Genetic Vectors ; genetics ; Glycosylation ; Pichia ; genetics ; metabolism ; Plasminogen Activators ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology