Solubilization and regeneration of Vitreoscilla hemoglobin isolated from protein inclusion bodies

Vitreoscilla hemoglobin (VHb), a homodimeric protein containing two heme groups in its native state, was used as a model to investigate inclusion body approtein solubilization, prosthetic group incorporation, and reactivation. High-level expression in recombinant Escherichia coli results in accumulation of a substantial portion of heme-free VHb in inclusion bodies. VHb can be solubilized from these inclusion bodies by relatively low concentrations of urea with the dissolution midpoint at approximately 3.2M urea. Dissolution in the presence of stoichiometric heme shifts the dissolution midpoint to approximately 4.5M urea without influencing the dissolution properties of contaminant proteins, suggesting the effect is specific for VHb. Denaturation of apoVHb and holoVHb obtained from purified native VHb has midpoints of 2.9M and 5.1M urea, respectively. VHb solubilized from inclusion bodies with urea at concentrations from 0 to 3.5M urea can be regenerated by heme addition without dilution of urea to yield active holoVHb. The fraction of solubilized VHb reconstituted upon heme addition is maximum at around 30% when solubilization and reconstitution is conducted in less than 1M urea. At these low urea concentrations, approximately 5% of inclusion body VHb is solubilized. These results show the utility of prosthetic group addition to reconstitute holoVHb in the presence of urea. Also, these findings suggest that some inclusion body protein has partially folded conformation and that a fractional dissolution and refolding process may be advantageous.     

Expression, purification, crystallization, and preliminary X-Ray analysis of the human UDP-glucose dehydrogenase

UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis of UDP-glucuronic acid from UDP-glucose resulting in the formation of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers. Here, human UGDH (hUGDH) was purified and crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data were collected to a resolution of 2.8 A. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 A, and alpha = beta = gamma = 90.0 degrees. Based on preliminary analysis of the diffraction data, we propose that the biological unit of hUGDH is a tetramer.     

Production of L-DOPA and dopamine in recombinant bacteria bearing the Vitreoscilla hemoglobin gene

Given the well-established beneficial effects of Vitreoscilla hemoglobin (VHb) on heterologous organisms, the potential of this protein for the production of L -DOPA and dopamine in two bacteria, Citrobacter freundii and Erwinia herbicola, was investigated. The constructed recombinants bearing the VHb gene (vgb⁺) had substantially higher levels of cytoplasmic L -DOPA (112 mg/L for C. freundii and 97 mg/L for E. herbicola) than their respective hosts (30.4 and 33.8 mg/L) and the vgb^– control strains (35.6 and 35.8 mg/L). Further, the vgb⁺ recombinants of C. freundii and E. herbicola had 20-fold and about two orders of magnitude higher dopamine levels than their hosts, repectively. The activity of tyrosine phenol-lyase, the enzyme converting L -tyrosine to L -DOPA, was well-correlated to cytoplasmic L -DOPA levels. As cultures aged, higher tyrosine phenol-lyase activity of the vgb⁺ strains was more apparent.     

透明颤菌血红蛋白基因vgb和腈水解酶基因bxn在毕赤酵母中的表达研究

为获得高效表达外源蛋白的Pichia pastoris菌株而设计了重组质粒pPIC9K—vgbbxn,其中透明颤菌血红蛋白基因vgb胞内表达以提高菌体的发酵密度,腈水解酶基因bxn分泌表达。转化GS115菌株后,通过PCR、SDS-PAGE检测证实两基因已经整合进酵母基因组且能高效表达,以及用准确的蛋白活性测定方法成功地检测到二所表达的产物均具有正常的活性。摇瓶发酵实验证明,血红蛋白在贫氧条件下可明显促进酵母菌体生长和bxn基因分泌表达。     

Expression,crystallization and preliminary X-ray diffraction study of FtsY,the docking protein of the signal recognition particle of E. coli

FtsY is the docking protein or SRα homologue in E. coli. It is involved in targeting secretory proteins to the cytoplasmic membrane by interacting with the signal recognition particle, controlled by guanosine 5′-triphosphate. Two different constructs have been used in crystallization studies: the full-length protein and a truncated fragment with a his-tag at the C terminus. Only the second construct resulted in crystals suitable for x-ray diffraction. The crystals belong to the monoclinic space group P2₁ with cell dimensions a = 32.20 Å, b = 79.57 Å, c = 59.21 Å, and β = 94.45, and contain one molecule per asymmetric unit. At cryogenic temperatures the crystals diffract to a resolution limit of 2.5 Å by using a rotating anode, and beyond 1.8 Å by using synchrotron radiation. Proteins 28:285–288, 1997. © 1997 Wiley-Liss Inc.     

Expression,purification, crystallization,and diffraction analysis of a selenomethionyl lipase Lip8 from Yarrowia lipolytica

Yarrowia lipolytica is a nonconventional model micro-organism with multiple biotechnological applications. It is also considered to be an excellent producer for lipase. Genome survey shows that Y. lipolytica possesses various paralogs of genes coding for extracellular, cell-bound, and intracellular lipolytic enzymes. However, little structural information on these isoenzymes is available. With the aim to facilitate crystal structure solution of Lip8, one of the most valuable lipases from Y. lipolytica, a less conventional protein expression technique—selenomethionyl protein expression was used to produce recombinant selenomethionine (SeMet)-Lip8 in Escherichia coli. Finally, three Met residues of Lip8 were all substituted with SeMet. A total of 72?mg of SeMet-Lip8 was obtained from a liter of the SeMet medium. Using sodium acetate as a precipitant and ammonium sulfate as an additive, crystals of the SeMet-Lip8 with 1.9?Å were successfully cultured through hanging-drop vapor diffusion method. The estimated crystal dimensions were 0.11?×?0.11?×?0.14?mm². The crystal belonged to the space group I4 with unit cell parameters a?=?b?=?128.87?Å, c?=?171.77?Å, α?=?β?=?γ?=?90°. It is the second member of lipase crystal family from Y. lipolytica. This work will provide a platform for further studying lipases from a structural insight.     

Expression,purification, and crystallization of meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum

The gene encoding the meso-diaminopimelate dehydrogenase (DAPDH) from Corynebacterium glutamicum was overexpressed and purified to homogeneity. Crystals of the binary DAPDH-NADP⁺ complex were obtained from solutions of polyethylene glycol 8000, 100 mM sodium cacodylate, pH 6.5, and 150–300 mM Mg(OAc)₂. The crystals diffract to 2.2 Å, belong to the orthorhombic space group P2₁, and contain two molecules per asymmetric unit. © 1996 Wiley-Liss, Inc.     

Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen. Journal of Industrial Microbiology & Biotechnology (2001) 27, 27–33. Received 20 October 2000/ Accepted in revised form 04 May 2001     

从透明颤菌血红蛋白谈到植物缺氧与转基因作物

扼要介绍透明颤菌血红蛋白基因在多种微生物的表达、调节和生理功能,特别是在生物工程方面可能的应用。但最值得重视的是这一基因在烟草中的表达及其生理效应,它为我们提出一个重要的问题,就是植物是否缺氧,透明颤菌血红蛋白基因很可能是构建转基因作物的重要元件。     

Epitaxial crystallization of alkane chain lipids for electron diffraction analysis

Thin microcrystals of a wide variety of polymethylene chain materials, including n-alkanes, linear waxes, glycerides, a detergent, phospholipids and phospholipid analogs based on cyclopentane-1,2,3-triol, are epitaxially grown on naphthalene to give an orientation with long chain axes parallel to the best developed crystal face. These crystals, which represent a different orientation than those grown from solution, facilitate ab initio quantitative crystal structure analysis from electron diffraction intensity data from the projection yielding the most crystallographic information.     

Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: New insights from NAD(P)H fluorescence and culture redox potential

On-line NAD(P)H fluorescence and culture redox potential (CRP) measurements were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia coli Batch cultures of a VHb-synthesizing E. coli strain and the iso-genic control under fully aerated conditions were subject to several high/low oxygen transitions, and the NAD(P)H fluorescence and CRP were monitored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4-fold under diminishing oxygen tension. In the absence of aeration, the strain producing VHb maintained a steady NAD(P)H level 1.8-fold less than that of the control, indicating that the presence of VHb keeps E. coli in a more oxidized state under oxygen-limited conditions. Estimated from CRP, the oxygen uptake rates near anoxia were 25% higher for cells with VHb than those without. These results suggest that VHb-expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utilization of VHb-expressing cells responds to rapidly changing oxygen tension, which is common in large-scale fermentations, we pulsed air intermittently into a cell suspension and recorded the fluorescence response to the imposed dissolved oxygen (DO) fluctuation. Relative to the control, cells containing VHb had a sluggish fluorescence response to sudden changes of oxygen tension, suggesting that VHb buffers intracellular redox perturbations caused by extracellular DO fluctuations.(c) John Wiley & Sons, Inc.     

Shedding light on the role of Vitreoscilla hemoglobin on cellular catabolic regulation by proteomic analysis

Heterologous expression of Vitreoscilla hemoglobin (VHb) has been reported to improve cell growth, protein synthesis, metabolite productivity and nitric oxide detoxification. Although it has been proposed that such phenomenon is attributed to the enhancement of respiration and energy metabolism by facilitating oxygen delivery, the mechanism of VHb action remains to be elucidated. In the present study, changes of protein expression profile in Escherichia coli as a consequence of VHb production was investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting. Total protein extracts derived from cells expressing native green fluorescent protein (GFPuv) and chimeric VHbGFPuv grown in Luria-Bertani broth were prepared by sonic disintegration. One hundred microgram of proteins was individually electrophoresed in IEF-agarose rod gels followed by gradient SDS-PAGE gels. Protein spots were excised from the gels, digested to peptide fragments by trypsin, and analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Results revealed that expression of VHbGFPuv caused an entire disappearance of tryptophanase as well as down-regulated proteins involved in various metabolic pathways, e.g. glycerol kinase, isocitrate dehydrogenase, aldehyde dehydrogenase, and D-glucose-D-galactose binding protein. Phenotypic assay of cellular indole production confirmed the differentially expressed tryptophanase enzymes in which cells expressing chimeric VHbGFP demonstrated a complete indole-negative reaction. Supplementation of delta-aminolevulinic acid (ALA) to the culture medium enhanced expression of glyceraldehyde-3-phosphate dehydrogenase and glycerol kinase. Our findings herein shed light on the functional roles of VHb on cellular carbon and nitrogen consumptions as well as regulation of other metabolic pathway intermediates, possibly by autoregulation of the catabolite repressor regulons.     

Fusion protein of Vitreoscilla hemoglobin with D-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C

In this study we constructed an artificial flavohemoprotein by fusing Vitreoscilla hemoglobin (VHb) with D-amino acid oxidase (DAO) of Rhodotorula gracilis to determine whether bacterial hemoglobin can be used as an oxygen donor to immobilized flavoenzyme. This chimeric enzyme significantly enhanced DAO activity and stability in the bioconversion process of cephalosporin C. In a 200-mL bioreactor, the catalytic efficiency of immobilized VHb-DAO against cephalosporin C was 12.5-fold higher than that of immobilized DAO, and the operational stability of the immobilized VHb-DAO was approximately threefold better than that of the immobilized DAO. In the scaled-up bioprocess with a 5-L bioreactor, immobilized VHb-DAO (2500 U/L) resulted in 99% bioconversion of 120 mM cephalosporin C within 60 min at an oxygen flow rate of 0.2 (v/v) x min. Ninety percent of the initial activity of immobilized VHb-DAO could be maintained at up to 50 cycles of the enzymatic reaction without exogenous addition of H(2)O(2) and flavin adenine dinucleotide (FAD). The purity of the final product, glutaryl-7-aminocephalosporanic acid, was confirmed to be 99.77% by high-performance liquid chromatography (HPLC) analysis. Relative specificity of immobilized VHb-DAO on D-alpha-aminoadipic acid, a precursor in cephalosporin C biosynthesis, increased twofold, compared with that of immobilized DAO, suggesting that conformational modification of the VHb-DAO fusion protein may be altered in favor of cephalosporin C.     

Purification,crystallization, and preliminary X-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis

The tRNA modifying enzyme tRNA–guanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the queuine precursor 7-aminomethyl-7-deazaguanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for x-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 Å, b = 65.1 Å, c = 71.9 Å, and β = 97.5°, and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 Å resolution.     

Expression, purification, crystallization and preliminary x-ray analysis of Cyan fluorescent protein CyPet

The technique of fluorescence (or F?rster) resonance energy transfer (FRET) is widely used to observe bimolecular interaction in living cells. Cyan and yellow fluorescent proteins are the most widely used pair in FRET analysis. CyPet and YPet are two newly optimized fluorescent proteins that have much better dynamic range and sensitivity than CFP/YFP pair, although the crystallographic structure and the mechanism of better fluorescent characteristics of CyPet are still unknown. We have expressed the cyan fluorescent protein CyPet using pT7 prokaryocyte expression system in Escherichia coli strain Rosetta (DE3) pLysS by auto-induction. After purification, the recombinant CyPet protein was crystallized by hanging drop vapor diffusion technique and could diffract to 2.55A resolution. The data showed that the orthorhombic CyPet crystal was in space group P212121 with unit cell parameters (51.55, 61.53, 63.36) and contained one molecule in one asymmetric unit.     

鼠脑驱动蛋白的表达、纯化和结晶

驱动蛋白是一类利用水解ATP为ADP和磷酸的过程中释放的能量沿微管系统运动的蛋白。为了研究ATP中储存的化学能是如何转化为驱动蛋白的机械动能,鼠脑驱动蛋白的相关N-端区域在BL21-Codon Plus(DE3)-RP感受态大肠杆菌细胞中大量地表达。通过SP-强阳离子交换色谱和分子筛色谱的两步骤纯化,蛋白最终产量高达10 mg/L细胞培养液,蛋白纯度可以达到95%以上。纯化的蛋白具有水解ATP酶的活力,并与驱动蛋白抗体有特异性的反应。驱动蛋白可以在如下条件结晶:1.7 mol/L(NH4)2SO4,500 mmol/L NaCl,20%glycerol。晶体衍射的分辨率可以达到2.0。     

斑贝链霉菌接合转移系统的构建及透明颤菌血红蛋白基因的表达对其次级代谢的影响

以pSET152和pHZ1358为出发质粒,通过供体大肠杆菌ET12567(pUZ8002)和S17-1接合转移斑贝链霉菌(Streptomycesbambergiensis),构建和优化了接合转移体系。采用OOE-PCR技术构建含ermEp*与vhb结构基因融合片段的整合型表达载体pBIB2005,转化ET12567(pUZ8002)后,属间接合转移至斑贝链霉菌。通过PCR、CO结合差光谱验证vhb基因在斑贝链霉菌中整合表达。摇瓶发酵显示VHb蛋白能改善菌体对氧的需求,一定程度上促进细胞生长,提高抗生素产量。     

Expression,purification, crystallization,and preliminary X-ray analysis of recombinant human saposin B

Saposin B (also known as cerebroside sulfate activator or CSAct) is a small non-enzymatic glycoprotein required for the breakdown of cerebroside sulfates (sulfatides) in lysosomes. Saposin B contains three intramolecular disulfide bridges, exists as a dimer and is remarkably heat, protease, and pH stable. We have expressed the protein in a thioredoxin reductase deficient strain of Escherichia coli and purified the protein by heat treatment, followed by ion-exchange, gel filtration, and hydrophobic interaction chromatographies. The protein is properly folded as judged by the observed disulfide bond topology, the hydrogen-deuterium exchange rate, and the level of stimulation of sulfatide hydrolysis by arylsulfatase A. Crystals of human saposin B were grown by vapor diffusion and diffract to a resolution of 2.2A. Despite obtaining only merohedrally twinned P3(1) native crystals, an untwined seleomethionine-substituted crystal belonging to space group P3(1)21 was also grown. The three-dimensional structure of saposin B protein will provide insights into how this 79 amino acid protein is able to solubilize relatively large membrane-bound lipid ligands.