Spore coat proteins, such as CotB, CotC, CotG et al, are able to efficiently display exogenous protein on spore surface for preparing oral vaccines or enzymes. CotX is another structural protein of spore coats of Bacillus subtilis. To investigate whether CotX could carry target protein onto the spore surface, we constructed a recombinant integrative plasmid, designated as pJS749, which carries a recombinant cotX-gfp gene under the control of the cotX promoter. We transformed pJS749 into Bacillus subtilis 168(trp-), an alpha-amylase inactivated mutant DRJS749 was selected and confirmed to be a double crossover integrant, where cotX-gfp fragment was integrated into the chromosome. After induction of spore formation, significant green fluorescence was observed on spore surface of strain DRJS749 under fluorescent microcopy. This suggests that CotX is associated with the outer part of the coat. CotX can therefore be used as a molecular vehicle for spore surface display of exogenous proteins.
Bacillus subtilis ; genetics ; metabolism ; physiology ; Bacterial Proteins ; biosynthesis ; genetics ; Gene Expression Regulation, Bacterial ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Spores, Bacterial ; genetics ; metabolism

For studying the effects of Mycobacterium tuberculosis secretory protein ESAT-6 on the related functions of macrophages, RAW264.7 cells were transfected with pEGFP-C1-ESAT-6 and pEGFP-C1 by liposome respectively. After screening with a high level of G418, the macrophage cell lines that stably expressed EGFP-ESAT-6 fusion protein or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and Western blotting. The results indicated that the EGFP-ESAT6 fusion gene was integrated into the chromosome and the protein could be stably expressed in the selected macrophage cell line. These results gave us a tool for the future study in the mechanisms of ESAT-6 protein in modulating the macrophage cells.
Animals ; Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Cell Line ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Macrophages ; cytology ; metabolism ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

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

This study was conducted to amplify the cfpl0-esat6 fusion gene by SOE and insert into the integrating shuttle plasmid pMV361 to form the recombinant plasmid. Then another recombinant plasmid was constructed by insertinga-A g signal sequence of BCG. The two recombinant plasmids were introduced into BCG and the induced products from recombinant BCG were analyzed. In conclusion,the successful construction of rBCG expressing the fusion protein CFP10-ESAT6 will be the base of the development of novel Mycobacterium tuberclosis vaccines.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Mycobacterium bovis ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Tuberculosis Vaccines ; biosynthesis

We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.
Bacterial Proteins ; biosynthesis ; genetics ; Catalytic Domain ; Cysteine Endopeptidases ; biosynthesis ; genetics ; Endopeptidases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Luminescent Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; SUMO-1 Protein ; biosynthesis ; genetics

Production of chiral amines and unnatural amino-acid using ω-transaminase can be achieved by kinetic resolution and asymmetric synthesis, thus ω-transaminase is of great importance in the synthesis of pharmaceutical intermediates. By genomic data mining, a putative ω-transaminase gene hbp was found in Burkholderia phytofirmans PsJN. The gene was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme (HBP) was purified by Ni-NTA column and its catalytic properties and substrate profile were studied. HBP showed high relative activity (33.80 U/mg) and enantioselectivity toward β-phenylalanine (β-Phe). The optimal reaction temperature and pH were 40 ℃ and 8.0-8.5, respectively. We also established a simpler and more effective method to detect the deamination reaction of β-Phe by UV absorption method using microplate reader, and demonstrated the thermodynamic property of this reaction. The substrate profiling showed that HBP was specific to β-Phe and its derivatives as the amino donor. HBP catalyzed the resolution of rac-β-Phe and its derivatives, the products (R)-amino acids were obtained with about 50% conversions and 99% ee.
Bacterial Proteins ; biosynthesis ; genetics ; Burkholderia ; enzymology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Transaminases ; biosynthesis ; genetics

To begin with, we constructed cfp10-esat6 fusion gene and its prokaryotic expression vector and had it express in E. coli. By GeneSOEing techniques, a fusion gene was constructed by splicing cfpl0 gene and esat6 gene, and then was cloned into pGEX-4T-1 plasmid. Secondly, we constructed the prokaryotic expression recombinant plasmid pGcfp10-esat6. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis, The E. coli BL21 containing the recombinant plasmid was induced by IPTG (Isopropy-beta-D-thiogalatoside). The fusion protein CFP10-ESAT6 with GST-tag about 42 kDa was expressed and purified with GST-fusion protein purification kit,The expression of cfp10-esat6 fusion gene was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The sequence of cfp10 and esat6 in recombinant plasmid was consistent with that of GenBank report. The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E. coil. The fusion protein was purified and the purity reached 90%. Its antigenicity was confirmed by Western-blotting. The prokaryotic expression vector (pGcfp1o-esat6) was constructed successfully, and the fusion protein CFP10-ESAT6 was obtained. This study provided an experimental basis for potential application of the recombinant CFP10-ESAT6 in the diagnosis of tuberculosis.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo construct recombinant BCG against leptospirosis.

METHODSWe amplified the entire open reading frame of the OmpL1 gene from the genome of the leptospire serovar Lai strain 017. Two recombinant plasmids pBQ1 and pBQ2 were constructed by oriented ligation based on the E. coli-BCG shuttle plasmids pMV261 and pMV361 respectively. The recombinant plasmids were transformed into BCG by electroporation. The rBCGs bearing pBQ1 and pBQ2 were induced by high temperature of 45 degrees C.

RESULTSThe expressed product, a 35kD protein was detected by SDS-PAGE. The result indicates that pBQ1 and pBQ2 can express OmpL1 in rBCG.

CONCLUSIONThe technical methods in this study may help detect the immunogenicity and immunoprotection of OmpL1 and develop more safe, highly effective rBCG bearing leptospiral antigen with long-lasting protection.

BCG Vaccine ; genetics ; Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; DNA, Bacterial ; genetics ; Gene Expression ; Genes, Bacterial ; Leptospira interrogans ; genetics ; Open Reading Frames ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis

Studies on lycopene synthesis in Escherichia coli were not only able to gain the strains with high yield and less by-products, but also able to test functions of genes or gene clusters. In this article, the cDNA sequences of tomato LeGGPS2 and LePSY1 as well as the coding sequence of crtI from Erwinia uredovora, each of which was added a ribosome biding site, were controlled by T7 promoter and terminator alone or combined, and expressed in E. coli BL21 (DE3) to induce lycopene synthesis. The results show that only T7::crtI-LeGGPS2-LePSY1 expressed tri-cistronically could produce lycopene, and 2.124 mg/g dry cell weight oflycopene was obtained when fermented for 5 h at 30 degrees C after mixing 80 micromol/L IPTG at the later logarithmic phase while the seed broth of 1:50 (V/V) was inoculated into LB medium (pH 6.8) containing 3% sucrose and cultured for 8 h at 37 degrees C. The results confirmed the function of the prokaryonized LeGGPS2 and LePSY1 and their synergy with crtI, and also laid a foundation to establish an independent lycopene synthetic pathway in the tomato plastid.
Bacterial Proteins ; genetics ; Carotenoids ; biosynthesis ; genetics ; Cloning, Molecular ; Erwinia ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Engineering ; Plant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Ergosterol is a principal sterol of fungi. It is a raw material for production of vitamin D2, hydrocortisone, progesterone and brassinolide. Synthesis of ergosterol requires molecular oxygen, and low oxygen tensions was reported to dramatically reduce ergosterol concentration. Vitreoscilla Hemoglobin Gene (vgb), a homodimeric hemoglobin gene from Gram-negative obligate aerobic bacterium Vitreoscilla, enables a higher specific cellular oxygen uptake rate, it also improves the oxygen transportation. In this study, recombinant plasmid pVgb-kanMX4 containing Vitreoscilla Hemoglobin Gene (vgb) and geneticin (G418) was constructed and transformed into Saccharomyces cerevisiae 1190 for enhanced ergosterol production. With sufficient oxygen supply, the ergosterol contents of recombinant and wild type strains grown in shake flasks were 1.07% and 0.573%, respectively. Under oxygen limitation condition, ergosterol contents in recombinant and wild type strains were reduced to 0.39% and 0.25%, respectively. In a 30 hours fermentation study conducted in a 5 liter fermentor, 15.1 g/L Cell Dry Weight (CDW) containing 1.38% ergosterol was obtained from growth of the recombinant strains; Only 14.8 g/L CDW containing 0.9% ergosterol was produced by the wild type strain. These results demonstrated that vgb played a role in enhancing ergosterol production.
Bacterial Proteins ; biosynthesis ; genetics ; physiology ; Cloning, Molecular ; Ergosterol ; biosynthesis ; genetics ; Fermentation ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Truncated Hemoglobins ; biosynthesis ; genetics ; physiology