Purification and Characterization of a DnaK-like and a GroEL-like Protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a ¹⁴C-labeled amino acid mixture was shifted from 37°C to 44°C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

Overexpression and purification of the galactose operon enzymes from Escherichia coli

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically cheracterized by gel-filtration column chromatography and isoelectric focusing, and the K_m values for their substrates were determined.     

Refolding and Characterization of a Yeast Dehydrodolichyl Diphosphate Synthase Overexpressed in Escherichia coli

Dehydrodolichyl diphosphate synthase (DDPPs) catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize long-chain dehydrodolichyl diphosphate, which serves as a precursor of glycosyl carrier in glycoprotein biosynthesis in eukaryotes. To perform kinetic and structural studies of DDPPs, we have expressed yeast DDPPs using Escherichia coli as the host cell. Thioredoxin and His tag were utilized to increase the solubility of the recombinant protein and facilitate its purification using Ni-nitrilotriacetic acid (NTA) column. The protein was overexpressed in E. coli but mostly existed in pellet in the absence of detergent. The low quantity of soluble DDPPs was purified using Ni-NTA, Mono Q anion-exchange, and size-column chromatographies. The protein in the pellet was solubilized with 7 M urea and purified using Ni-NTA under denaturing condition. The protein refolding was achieved via the stepwise dialysis to remove the denaturant in the presence of 6 mM β-mercaptoethanol. Detergent n-octyl-β- -glucopyranoside and Triton X-100 increased the solubility of the DDPPs so that refolding can be performed at higher protein concentration. Alternatively, on-column refolding was carried out in a single step to obtain the active protein in large quantities. β-Mercaptoethanol and Triton were both required in this quick refolding process. The kinetic studies indicated that the soluble and refolded DDPPs have comparable activities (k_cat = 2 × 10⁻⁴ s⁻¹). Unlike its bacterial homologue, undecaprenyl diphosphate synthase, yeast DDPPs activity was not enhanced by Triton.     

Refolding and Purification of Zymomonas mobilis Levansucrase Produced as Inclusion Bodies in Fed-Batch Culture of Recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE–Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Proteomics of Porphyromonas gingivalis within a model oral microbial community

Background  

Porphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community.     

Structural Study of Escherichia coli NAD Synthetase: Overexpression, Purification, Crystallization, and Preliminary Crystallographic Analysis

Escherichia coli NAD synthetase was overexpressed and purified to homogeneity. The recombinant protein was active in an in vitro enzyme assay. The enzyme required approximately 1.5 mM magnesium for optimal activity. The pH optimum was found to be 8.0-8.5. The recombinant protein was crystallized at room temperature using the hanging-drop vapor diffusion technique with 1.5 M lithium sulfate, 0. 1 M Hepes buffer at pH 7.5 as precipitant. The protein was also crystallized in the presence of its substrates, nicotinic acid adenine dinucleotide and adenosine triphosphate under similar conditions. These crystals diffract to 2.0-A resolution and belong to trigonal space group P3(1)21 with unit cell dimensions of a = b = 91.766, c = 74.17 A and alpha = beta = 90 degrees, gamma = 120 degrees. The structure of the complex has been determined using the molecular replacement method.     

Purification and characterization of the -xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli -xylose isomerase ( -xylose ketol-isomerase, EC 5.3.1.5) gene and the -xylulokinase (ATP: -xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The -xylose isomerase gene was separated from the -xylulokinase gene by the construction of a new deletion plasmid, pLX7. The -xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the -xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first -xylose isomerase gene that has been isolated and extensively purified from any organism. -Xylose isomerase, the enzyme product of the -xylose isomerase gene, is responsible for the conversion of -xylose to -xylulose, as well as -glucose to -fructose. It is widely believed that yeast cannot ferment -xylose to ethanol primarily because of the lack of -xylose isomerase in yeast. -Xylose isomerase (also known as -glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the -xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting -xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.     

Comparative transcriptomic analysis of Porphyromonas gingivalis biofilm and planktonic cells

Background  

Porphyromonas gingivalis in subgingival dental plaque, as part of a mature biofilm, has been strongly implicated in the onset and progression of chronic periodontitis. In this study using DNA microarray we compared the global gene expression of a P. gingivalis biofilm with that of its planktonic counterpart grown in the same continuous culture.     

Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions

We have developed an anaerobic biofilm culture system. The system is inexpensive, simple to use and, unlike an anaerobic glovebox, requires no dedicated space. As a test of the system, Porphyromonas gingivalis was cultured under low oxygen (1–2 ppm) and under anaerobic conditions (≤0.1 ppm O₂). In the presence of small amounts of oxygen, the organism attached and formed an initial biofilm over the course of 4 h, but the biofilm was unable to maintain its growth and had lost biomass after 18 h. Also, ambiguous results were obtained when the biofilm was stained with a viability stain. Under anaerobic conditions, the biofilm was able to continue growth — biomass was greater after 18 h than after 4 h, and the anaerobic biofilm had a less ambiguous staining pattern than did the low-O₂-grown biofilm.     

产GL-7-ACA酰化酶重组菌的构建及高表达研究

戊二酰基-7-氨基头孢烷酸(GL-7-ACA)酰化酶是7-氨基头孢烷酸(7-ACA)两步酶法生产中的关键酶。成功构建组成型表达的产GL-7-ACA酰化酶重组大肠杆菌JM105/pMKC-ACY,并对其高表达条件进行了研究,得到了组成简单、廉价的国产培养基配方及操作简便、易于实现工业化的发酵工艺。在优化条件下,上罐补料高密度发酵的酶活高达6668.9U/L,是优化前的12.4倍,产率最高可达275.5U/(L.h),达到了工业生产的要求。     

Overexpression in Escherichia coli and Characterization of the Chloroplast Triosephosphate Isomerase from Spinach

An important Calvin cycle enzyme, chloroplast triosephosphate isomerase (cpTPI) from spinach, has been cloned and expressed in up to 15% of the total cell protein using the P(L) expression vector in Escherichia coli. An even higher level expression, up to 36% of the total protein, was achieved by replacing the nucleotide sequence between the ribosomal binding site and the initial codon, ATG, with an AT-rich sequence. Computer modeling revealed that the moderate change in the standard free energy (5'-DeltaG degrees ) of mRNA secondary structure in the translation initial region might be the major factor which led to the later high-level expression. The overexpressed spinach cpTPI was soluble and fully active and was able to be purified beyond 95% purity by DEAE-Sepharose and Sephadex G-75, and around 55 mg of purified enzymes was obtained from 1 liter of cultured bacteria. With d-glyceraldehyde 3-phosphate as substrate, K(m (D-3-P)) is 0. 68 mM, V(max (G-3-P)) is 3.16 x 10(4) micromol/min. mg, and K(cat (G-3-P)) is 4.51 x 10(3)/s; with dihydroxyacetone phosphate as substrate, the corresponding values are 7.27 mM, 1.04 x 10(3) micromol/min. mg, and 1.16 x 10(2)/s, respectively.     

Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

Cloning, Expression, and Purification of the Uropathogenic Escherichia coli Invasin DraD

In this study we presented a very efficient expression system, based on pET30LIC/Ek vector, for producing DraD invasin of the uropathogenic Escherichia coli and a one-step chromatography purification procedure for obtaining pure recombinant protein (DraD-C-His₆). This protein has a molecular weight of 14,818 and calculated pI of 6.6. It contains a polyhistidine tag at the C-terminus (13 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. Also, we obtained specific antibodies against DraD invasin to develop tools for characterizing the expression and biological function of this protein. The amount and quality of DraD-C-His₆ fusion protein purified from E. coli overexpression system seems to be fully appropriate for crystallographic studies (soluble form), and for establishing role of the protein in bacterium (cultured cell line interaction and in the internalization process) and for obtaining rabbit polyclonal antisera (insoluble form).     

Pathway analysis for intracellular Porphyromonas gingivalis using a strain ATCC 33277 specific database

Background  

Porphyromonas gingivalis is a Gram-negative intracellular pathogen associated with periodontal disease. We have previously reported on whole-cell quantitative proteomic analyses to investigate the differential expression of virulence factors as the organism transitions from an extracellular to intracellular lifestyle. The original results with the invasive strain P. gingivalis ATCC 33277 were obtained using the genome sequence available at the time, strain W83 [GenBank: AE015924]. We present here a re-processed dataset using the recently published genome annotation specific for strain ATCC 33277 [GenBank: AP009380] and an analysis of differential abundance based on metabolic pathways rather than individual proteins.     

大肠杆菌冷休克蛋白CspC的分离纯化及部分性质测定

经Sepharose Q Fast Flow阴离子交换层析和Superdex 30凝胶过滤层析,从大肠杆菌（Escherichia coli）细胞内分离纯化了一种小分子蛋白质，SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)纯度鉴定为单一条带，经质谱分析、N端测序、同源序列比较，确定该蛋白质为大肠杆菌冷休克蛋白CspC.在此基础上，用圆二色光谱测定了其二级结构含量，初步探索了其热稳定性及与单链DNA结合后的构象变化.     

Morphogenesis of Escherichia coli

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

毕赤酵母甲酸脱氢酶在大肠杆菌中的高表达及纯化

用PCR方法从毕赤酵母(Pichia pastoris)基因组DNA扩增甲酸脱氢酶(FDH)基因,通过定点突变密码子TAG(649-651位碱基)为GAG,突变后的基因片段插入表达载体pET-22b( )构建质粒pET-FDH,转化E．coli BL21(DE3)。基因工程菌在IPTG诱导下高效表达可溶性的FDH融合蛋白,蛋白的表达量占基因工程菌株总蛋白的30%。工程菌破壁上清液采用一步亲和层析分离,得到比活力为6．45U/mg的重组FDH。     

Cloning, Overexpression, Refolding, and Purification of the Nonspecific Phospholipase C fromBacillus cereus

Bacillus cereussecretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester.B. cereusPLC has been overexpressed with its signal sequence inEscherichia coliusing a T7 expression system. The expressed enzyme formed intracellular inclusion bodies which were solubilized in the presence of 8 urea. Renaturation was initiated by gradual removal of urea and addition of zinc ions. The signal peptide was specifically cleaved by a protease, clostripain, added when the urea concentration was 1.5 . Factors that led to protein reaggregation included rapid removal of urea, use of Tris instead of barbital buffer, and presence of the signal peptide when the urea concentration was below 1.5 . The folded protein was purified by Q-Sepharose Fast Flow chromatography to yield a preparation >99% pure. The final yield of active enzyme was 30–40 mg per liter of culture. The recombinant PLC exhibited biochemical and kinetic properties identical to those of extracellularly produced PLC fromB. cereus.Site-specific mutagenesis of Asn-134 was carried out as a test of the general effectiveness of the refolding procedure.     

Escherichia coli and its chromosome

The Escherichia coli chromosome is a circular DNA molecule that is approximately 1000 times compacted in the living cell, where it occupies approximately 15% of the cellular volume. The genome is organized in a way that facilitates chromosome maintenance and processing. Despite huge efforts, until recently little has been known about how the chromosome is organized within cells, where replication takes place, and how DNA is segregated before cell division. New techniques for labeling genetic loci and molecular machines are allowing the simultaneous tracking of genetic loci and such machines in living cells over time. These studies reveal remarkable organization, yet a highly dynamic flux of genetic loci and macromolecules. It seems likely that the cellular positioning of chromosomal loci is the outcome of the formation of two chromosome arms (replichores) by replication, followed by sequential chromosome segregation, rather than from the presence of cellular positioning markers.