Figure 1 The effect of trifluorothymidine (TFT) on the uptake of [ 3 H]-dT (●), TK (■) and TS (▲) activity. Mpn wild type cells were cultured in the find more presence of [3H]-dT and different concentrations of TFT. The cells were incubated at 37°C for 70 hours and harvested. The total uptake and incorporation of [3H]-dT were analysed, and TK and TS activity were determined in total protein extracts. Expression, purification, and characterization of HPRT The purine analog 6-TG strongly inhibited Mpn growth, which promoted further investigation of potential targets of this compound. HPRT is the first enzyme in the salvage pathway of purine bases

for nucleotide biosynthesis, and is the enzyme responsible for metabolizing 6-TG in human patients treated with this

drug [37]. Mpn HPRT (MPN672) consists of 175 amino acids and shares 29% sequence identity to human HPRT. Mpn HPRT cDNA was cloned and expressed in E. coli. Recombinant Mpn HPRT was expressed as an N-terminal fusion protein with a 6 × histidine tag and a tobacco etch virus (TEV) cleavage site at the N-terminus, and was purified to >98% purity by metal affinity chromatography, see more as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (data not shown). The purified Mpn HPRT used both hypoxanthine (Hx) and guanine (Gua) as substrates but not adenine or uracil. With Hx as substrate the Selleckchem ABT 263 reaction was linear with time for up to 25 min and the substrate saturation curve was hyperbolic, which indicated that the enzyme followed Michaelis–Menten kinetics with a Km value of 100.1 ± 6.5 μM and Vmax value of 15.8 ± 0.8 μmol min-1 mg-1 (Figure 2A). However, Dolutegravir nmr with Gua as a substrate, the reverse reaction rate was very high and the reaction reached equilibrium in less than 5 min under the same conditions used

for Hx. Therefore, the kinetic study with Gua was conducted differently as described in the experimental procedures. Substrate saturation for Gua exhibited a biphasic curve and therefore, data was fitted using the Hill equation. The Vmax value was 2.7 ± 0.1 μmol min-1 mg-1 and S0.5 was 107.6 ± 6.2 μM with a Hill coefficient of 3.5 (Figure 2B), indicating positive cooperativity with Gua binding. Figure 2 Substrate saturation curves of hypoxanthine (A) and guanine (B) with Mpn HPRT. Kinetic parameters for Hx and Gua were determined by using the DE81 filter paper assay with [3H]-Hx and [3H]-Gua as the labelled substrates as described in the experimental procedures. Data are from at least three independent measurements and are presented as mean ± standard deviation (SD).

Changes observed in proton leak, UCP expression, and circulating hormones appear to influence metabolic efficiency and energy expenditure. In the context of energy restriction, the observed changes are likely to make weight loss increasingly challenging and promote weight regain. It has been reported that females have more BAT than males [63], and that energy-restricted female rats see greater decreases in BAT mass and UCP-1 than males [64], indicating a potential sex-related difference in uncoupled respiration during weight loss. Subjects identified as “diet-resistant” show decreased proton leak and

UCP-3 expression compared to “diet-responsive” subjects during maintenance of a reduced bodyweight [65]. Brigatinib solubility dmso More research is needed to determine if these differential responses to hypocaloric diets

make sustained weight loss more difficult for females and certain predisposed “diet-resistant” individuals. While future research may improve our understanding of the magnitude and relative importance of mitochondrial adaptations to energy restriction, current evidence suggests that increased mitochondrial efficiency, and a decline in uncoupled respiration, might serve to decrease the energy deficit in hypocaloric conditions, making weight maintenance and further weight reduction more challenging. Practical applications for weight loss in athletes Hypocaloric diets induce a number of adaptations that serve to prevent further weight loss and conserve energy. It is likely that the magnitude of these adaptations are proportional to the size of the energy deficit, so it is recommended see more to utilize the smallest possible deficit that yields appreciable weight loss. This may decrease the rate of weight loss, but attenuate unfavorable adaptations that challenge successful reduction of fat mass. Weight reduction should be viewed as a stepwise process in this context; as weight loss begins to plateau, energy

intake or expenditure should not be adjusted to “re-open” the energy deficit. Large caloric deficits are also likely to induce greater losses of LBM [66, 67] and compromise athletic performance and recovery [68, 69], which are of critical importance to athletes. Participation in a structured resistance VX-689 mouse training program [34] and sufficient protein intake [35–37] are also likely to attenuate losses in LBM. Additionally, high protein diets (≥25%PRO) are associated with increased satiety and thermogenesis, making them a better option for the calorie-restricted athlete [70]. In the world of physique sports, periodic “refeeding” has become common in periods of extended dieting. A refeed consists of a brief overfeeding period in which caloric intake is raised slightly above maintenance levels, and the increase in caloric intake is predominantly achieved by increasing carbohydrate consumption.

Organic antibacterial agent has many disadvantages, including the toxicity hazard to the human body and instability in high temperature and pressure [10]. By comparison, inorganic antibacterial agent has the properties of heat resistance, long life, and chemical stability [11]. Nowadays, metallic simple substances and their compounds are used widely in

antimicrobial application research, such as Ag selleck chemicals [12–16], Fe2O3[17], TiO2[18], CuO [19, 20], MgO [21], Mg (OH)2[22], and ZnO [23, 24]. Among metal oxide antibacterial agents, ZnO has aroused concern due to its good antibacterial activities on a broad spectrum of bacteria [24–26]. The antibacterial properties of ZnO have been studied broadly with Selleckchem CUDC-907 pathogenic and nonpathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Klebsiella

pneumoniae, Pseudomonas, etc. [26, 27]. Zinc oxide is an interesting material due to its extensive applications in various areas, such as antibacterial, optical, piezoelectric, magnetic, and gas sensing properties [24, 26–31]. Therefore, many of the synthetic approaches such as sol-gel method [32], co-precipitation [31], hydrothermal method [33], microwave synthesis [23, 26], and thermal evaporation method [34] have been used for the preparation of ZnO powders. Hydrothermal method is an important technology in synthetic material. Using this method, the crystal grain can develop completely and the particle size is uniform. In this work, in order to research the influence of the microstructure and crystal on the Angiogenesis inhibitor antibacterial properties of titanium-doped ZnO powders, the powders were synthesized by alcohothermal method from different zinc salts, and the antibacterial activities against E. coli and S. aureus were evaluated. Moreover, the antibacterial mechanism Pregnenolone of titanium-doped

ZnO powders was deduced. Materials and methods Materials The reagents (e.g., two hydrated zinc acetate, zinc vitriol, zinc nitrate, zinc chloride, lithium hydroxide monohydrate, absolute ethyl alcohol, tetrabutyl titanate, glutaraldehyde, disodium hydrogen phosphate 12-hydrate, monopotassium phosphate) used in this study were analytically pure chemicals. Biological reagents (e.g., nutrient broth, nutrient agar medium) were used as received. De-ionized water and aquae sterilisata with conductivity lower than 0.5 μS/cm were used to prepare all the solutions. E. coli (ATCC44104) and S. aureus (CMCC26001) bacterial strains were obtained from Beijing Assay Institute of Biological Products. Phosphate-buffered saline (PBS; pH = 7.4) was prepared with disodium hydrogen phosphate 12-hydrate and monopotassium phosphate. Synthesis of titanium-doped ZnO powders Under magnetic stirring condition, 0.1 mol/L zinc salts and 0.14 mol/L lithium hydroxide alcoholic solution were prepared. Meanwhile, 0.01 mol/L tetrabutyl titanate alcoholic solution was prepared.

02 kg. Heart rate was determined by

POLAR® (Finland) heart rate monitor. Blood pressure was assessed in the supine position after resting for 5-min using a mercurial sphygmomanometer via standard procedures. Subjects then had body composition determined using hydrodensitometry BYL719 using standard procedures. Subjects reported to the Human Performance Lab in swimsuits and had their body weight determined out of water by an electronic scale. Body composition was analyzed using an EXERTECH (La Cresent, MN) body density measuring system that utilizes a weighing platform with electronic (load cell) weighing system connected to a PC. Calibration is conducted daily by establishing linear interpolation from 2 known weights. Data points were recorded with data acquisition software from the force transducer. Residual volume was estimated using standard procedures [18]. Subjects were submerged in warm water and asked to exhale a maximal amount of air while a signal from the force transducer produced a readable analog wave. The most stable waveform was selected, and the mean value was recorded. Subjects performed this procedure until at least 2 trials were within a 0.10% difference Pevonedistat or a total

of 7 trials were completed. Next, body density was calculated after weight was recorded in and out of water, and the Siri equation was used to calculate percentage of body fat [19]. Fat-free mass (FFM) was also calculated from the percentage of body fat [20]. Subjects then donated approximately 20 ml of fasting blood using buy RG-7388 venipuncture techniques of an antecubital Cell press vein in the forearm according to standard procedures. Blood samples were shipped to Quest Diagnostics (Dallas, TX) to run clinical chemistry profile, hepatic function, and whole blood cell counts. Blood samples were also centrifuged and aliquoted to microcentrifuge tubes and stored at -40°C for future analyses. Serum samples were then assayed in duplicate for the hormones free testosterone, Insulin, leptin, cortisol

(Diagnostics Systems Laboratories, Webster, TX), and dihydrotestosterone (DHT), estradiol (Alpco Diagnostics, Windham, NH), using enzyme-linked immunoabsorbent assays (ELISA) and enzyme-immunoabsorbent assays (EIA) using a Wallac Victor-1420 microplate reader (Perkin-Elmer Life Sciences, Boston, MA), and the assays were performed at a wavelength or either 450 or 405 nm, respectively in the Exercise and Biochemical Nutrition Lab at Baylor University. Subjects then performed 1 repetition maximum lifts (1-RM) on the isotonic bench press and leg press to assess strength and then muscular endurance. All strength/exercise tests were supervised by lab assistants experienced in conducting strength/anaerobic exercise tests using standard procedures. Subjects warmed-up (2 sets of 8 – 10 repetitions at approximately 50% of anticipated maximum) on the bench press.

Given the EPZ5676 General difficulty in defining bacterial species and the ready availability of genome sequence data,

we sought to evaluate a range of novel genotypic and genome-based metrics for species delineation. In light of discussed obstacles and the on-going public health concern, we believe that genus Acinetobacter provides a timely test case to evaluate the validity and robustness of these sequence-based approaches. In pursuit of this goal, we generated a diverse and informative set of thirteen new draft genome sequences, representing ten species, and we analyzed the whole-genome sequences from a total of 38 strains BI 2536 nmr belonging to the genus. Results and discussion General genome characteristics The genomes of thirteen Acinetobacter strains, including seven type strains, were sequenced to draft quality using 454 sequencing (Table 1). The A. bereziniae strain was found to have the largest genome size within the genus (~ 5 Mb), while the strain with the smallest genome (~2.9 Mb) belonged to the species A. parvus, which is known to have a reduced metabolic repertoire compared to see more other Acinetobacter species [39]. These thirteen genomes were considered

alongside twenty-five other publicly available genome sequences from the genus Acinetobacter (see Additional file 1). Table 1 Genome sizes, sequencing statistics, G+C content, number of CDSs in the thirteen sequenced Acinetobacter isolates   Species   Strain Genome size (Mb) Peak coverage No. of contigs G+C content (%) No. of predicted good quality CDSs† GenBank accession number A. parvus DSM 16617 (T) 2.88 24x 257 41.6 2681 AIEB00000000

A. radioresistens DSM 6976 (T) 3.35 13x 354 41.4 2964 AIDZ00000000 A. lwoffii NCTC 5866 (T) 3.35 14x 260 43.0 3005 AIEL00000000 A. ursingii DSM 16037 (T) 3.57 21x 158 40.0 3252 AIEA00000000 A. pittii* DSM 21653 (T) 3.75 8x 468 38.8 3252 AIEK00000000 A. calcoaceticus DSM 30006 (T) 3.89 10x 373 38.6 3377 AIEC00000000 A. baumannii W6976 3.91 8x 537 39.0 3252 Cyclin-dependent kinase 3 AIEG00000000 A. baumannii W7282 3.95 14x 140 39.0 3466 AIEH00000000 A. baumannii NCTC 7422 3.99 22x 179 41.3 3626 AIED00000000 A. pittii* DSM 9306 4.03 11x 339 38.8 3553 AIEF00000000 A. nosocomialis* NCTC 8102 4.12 10x 283 38.7 3596 AIEJ00000000 A. nosocomialis* NCTC 10304 4.16 10x 387 39.1 3501 AIEE00000000 A. bereziniae LMG 1003 (T) 4.98 12x 392 38.1 4480 AIEI00000000 * Species names as proposed by Nemec et al.[39]. † Definition of good quality CDS is length ≥ 50 codons, of which less than 2% are stop codons. (T) = Type strain. A. ursingii DSM 16037 genome characteristics The species A. ursingii was first described by Nemec et al. in 2001 [40]. We have genome sequenced the type strain DSM 16037, which was isolated from a blood culture taken from an inpatient in Prague, Czech Republic in 1993 [40].

The formed oxide covers Torin 2 all the internal surface of the porous nanowires and leads to expansion of the volume of the Si nanostructures composing the SiNW skeleton (Figure 3b). With the additional HF dip, the SiO2 layer from the internal porous Si surface is dissolved, selleck screening library leading to full dissolution of the upper length of the nanowires, which is highly porous (Figure 3c). This proves that the whole volume of the SiNWs is fully porous and that there is no single-crystal Si core

in the nanowires. This was an open question in the literature [11]. The fact that after the first HF/piranha treatment the length of the SiNWs is only slightly reduced, while after the additional HF dip the NWs www.selleckchem.com/products/tpx-0005.html almost disappear, except of a short nanowire base, indicates that the SiNW porosity is not homogeneous throughout their length, but it is higher at their top and it gradually decreases from the top to the bottom. In addition, the fact that the above chemical treatment did not dissolve the porous Si layer underneath the SiNWs means that the porosity of this layer is lower than that of the SiNWs’ tops. Consequently, in the as-grown sample, this layer is not expected to have

a significant contribution to the PL spectrum. Photoluminescence spectra PL spectra were obtained from the as-formed samples and from samples after different chemical treatments. PL was excited by a HeCd laser line at 325 nm. The results are summarized in Figure 4 for a sample etched for 60 min. The PL peak is broad, with a maximum at approximately 1.9 eV and a full width at half maximum (FWHM) of approximately 380 meV in the case of the as-formed sample. By immersing the as-etched sample into an HF solution, the PL peak was red-shifted from 1.73 to 1.80 eV while the PL FWHM increased from 412 to 447 meV. In addition, the PL intensity increased by a factor of 2. The HF dip was then followed by a piranha treatment that oxidizes the internal Si surface, forming an oxide shell around the nanostructures composing

the porous nanowire skeleton. This treatment old caused a shift of the PL wavelength to approximately the initial peak energy and the initial FWHM. In addition, the PL intensity was doubled. Finally, after an additional HF treatment, the PL intensity was increased by 50 times, without any significant wavelength shift. These results will be discussed below. Figure 4 PL spectra from the as-grown sample etched for 60 min and samples after different chemical treatments. The spectrum from the as-grown sample is denoted by (1), the sample after an HF dip by (2), after HF/piranha by (3), and after HF/piranha/HF by (4). The vertical dashed line is a guide to the eye. From time-resolved PL measurements, the PL decay time at room temperature was found to be in the 19- to 23-μs range.

Nature 2000, 406:959–964.PubMedCrossRef 17. Parret AHA, De Mot R: Bacteria killing their own kind, novel bacteriocins of Pseudomona and other gamma-proteobacteria. Trends Microbiol 2002, 10:107–112.PubMedCrossRef 18. Waite RD, Curtis MA: Pseudomonas aeruginos PAO1 pyocin production affects population dynamics within mixed-culture biofilms. J Bacteriol 2009, 191:1349–1354.PubMedCrossRef 19. Köhler T, Donner

V, van Delden C: Lipopolysaccharide as shield and receptor for R-pyocin-mediated killing in Pseudomonas aeruginos . J Bacteriol 2010, 192:1921–1928.PubMedCrossRef 20. De Jong A, Van Hijum SAFT, Bijlsma JJE, Kok J, Kuipers OP: BAGEL, a web-based bacteriocin genome mining tool. Nucleic Acids Res 2006, 34:W273-W279.PubMedCrossRef BI 10773 concentration 21. Bourke WJ, O’Connor CM, FitzGerald MX, McDonnell TJ: Pseudomonas aeruginos exotoxin A Selleck PF299804 induces pulmonary endothelial cytotoxicity: protection by dibutyryl-cAMP. Eur Respir J 1994, 7:1754–1758.PubMedCrossRef 22. Caldwell CC, Chen Y, Goetzmann HS,

Hao Y, Borchers MT, et al.: Pseudomonas aeruginosa exotoxin pyocyanin causes cystic fibrosis airway pathogenesis. Am J Pathol 2009, 175:2473–2488.PubMedCrossRef 23. Kudurugamuwa JL, Beveridge TJ: Bacteriolytic effect of membranve vesicles from Pseudomonas aeruginos on other bacteria including pathogens: conceptually new antibiotics. J Bacteriol 1996, 178:2767–2774. 24. Aaron SD, Vandemheen KL, Ramotar see more K, Giesbrecht-Lewis T, Tullis E, et al.: Infection with transmissible strains of Pseudomonas aeruginos and clinical outcomes in adults with cystic

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Authors’ contributions RD and DF carried out the laboratory experiments. MC and MCT designed the study and RD, MC and MCT wrote the manuscript. All authors read and approved the final manuscript.”
“Background Eukaryotic genomes are packaged into the nucleus by histones and non histone proteins. Histones are small, highly basic proteins that form a core around which the DNA is wrapped. Although chromatin is highly compacted, its structure is dynamic, allowing access to the DNA for processes such as replication, transcription, Selisistat recombination and repair [1, 2]. Nucleoid-associated proteins have been described in archaea and bacteria. These proteins resemble eukaryotic histones in their DNA binding properties,

low molecular weight, abundance and electrostatic charge. They organize and compact the prokaryotic genome and are involved in various processes, including gene expression [3, 4]. The proteins involved in DNA packaging in eukaryotic organelles have check details not been fully characterized. In the protozoa of the Trypanosomatidae family, the mitochondrial genome is contained within a specific region of the mitochondrion known as the kinetoplast. The kinetoplast DNA (kDNA) of trypanosomatids is organized into an unusual arrangement of circular molecules, catenated into a single network. Two types of DNA ring are present within the kinetoplast: maxicircles and minicircles. The maxicircles resemble the mitochondrial DNA of higher SRT1720 order eukaryotes, encoding rRNAs and subunits of the respiratory complexes [5]. The minicircles

encode guide RNAs, which modify the maxicircle transcripts by extensive insertions and/or deletions of uridylate residues to form functional open reading frames, in a process known as RNA editing [6]. The replication of kinetoplast DNA requires a repertoire of molecules, including type II topoisomerases, Thalidomide DNA polymerases, universal minicircle sequence binding proteins, primases and ribonucleases [7, 8]. The molecules involved in maintaining the highly ordered organization of kDNA in trypanosomatids remained unknown for many years. In 1965, Steinert suggested that the kinetoplast DNA was not associated with basic proteins [9]. However, Souto-Padrón and De Souza provided cytochemical evidence that the kDNA of Trypanosoma cruzi was associated with basic proteins [10, 11]. They suggested that such proteins might be involved in neutralizing the negatively charged DNA molecules in close contact within the kinetoplast matrix.

h Maissau Arable field ITS/LSU 96 19 20.4 ± 3.1 92.8 2.33 7.37 Niederschleinz Arable field ITS/LSU 92 34 51.3 ± 12.0 Alvocidib order 66.3 3.27 28.09 Purkersdorf Arable field ITS/LSU 94 32 44.9 ± 9.5 71.3 3.18 23.76 Riederberg Grassland ITS/LSU 92 31 41.4 ± 7.1 77.3 2.84 10.76 Tulln Arable field ITS/LSU 89 24 32.9 ± 8.0 72.9 2.84 15.48 Sourhope (UK)a Grassland SSU 53 18 47.8 ± 22.4 37.7 1.93 3.62 Sourhope (UK)a Grassland ITS 45 22 51.3 ± 20.5 42.9 2.53 7.50 Cristalina (BRA)a Arable field (Soy) SSU 104 22 30.9 ± 7.6 71.2 1.87 2.87 aData for the soils “Sourhope” from the Sourhope Research Station in Scotland, UK (Anderson et al. 2003) and “Cristalina” from the district Cristalina in Goiás, Brazil (de Castro et al. 2008) were taken from the respective publications

bLibrary indicates on which region from rRNA-encoding cluster profiling of the fungal community was done cClones: number of INCB018424 supplier analysed clones for each soil; dSobs: number of observed species in the clone libraries; eChao2 ± SD: Estimated species richness ± standard deviation for the sampling site https://www.selleckchem.com/products/S31-201.html based on the Chao2 richness estimator (Chao 1987) implemented in EstimateS 8.2; f% Cov.: Estimated coverage of the libraries based on observed and estimated species richness; gShann.: Shannon Diversity Index hSimp.: Simpson Diversity Index UniFrac was used to compare the phylogenetic structures of the fungal communities from soils M, N, P, R and T (Lozupone

et al. 2006). To this end sequences were aligned with the ClustalW algorithm in MEGA4 (Tamura et al. 2007), and a neighbor-joining tree was

calculated from the aligned partial LSU sequences. The ITS-region was excluded, since it cannot be unambiguously aligned over such a broad phylogenetic distance. Sequences from an unknown eukaryote (NG_R_F10, Acc. Nr. GU055695) and from a fungus of uncertain affiliation (NG_R_F02, Acc Nr. GU055690) from site R were used as outgroups and excluded from further analyses. Data were weighted for abundance and normalized for branch length for calculating the UniFrac metric of the distance between each pair of soil samples (Lozupone et al. 2006). Results Soil characteristics of the five soils used in the present study are given in Inselsbacher et al. (2009). All soil parameters are within the range for typical arable land as used for cultivation of barley in this area. Fungal communities were Celastrol analysed by direct amplification of fungal ITS/partial LSU regions with primer pair ITS1F and TW13. Cloned PCR products from each soil were grouped by RFLP and up to four representatives from each RFLP type were sequenced. By this approach even closely related sequences (e.g. four different Tetracladium species from soil P with a maximum sequence difference of 3.7%) could be dissected. While the ITS region provides excellent resolution down to the species level, the partial LSU region provides good resolution at higher taxonomic levels when sufficiently identified ITS reference data in public databases are missing (Urban et al. 2008).

Based on sequence analysis, VirB1-89K was predicted to contain a C-terminal CHAP domain (located between the amino acids 796 and 926) and an N-terminal transmembrane domain, but lacks a signal sequence. The CHAP domain is broadly found in proteins from bacteria, phages, archaea, and eukaryotes of the Trypanosomidae family [19, 20]. It has been proposed that the CHAP domain may function mainly in peptidoglycan hydrolysis [19]. The phylogenetic analysis of VirB1-89K and its homologous proteins showed that VirB1-89K and N-acetylmuramoyl-L-alanine amidase probably originate from the same ancestor (Figure 1A). Figure 1 Sequence

analysis of VirB1-89K. (A) Phylogenetic analysis of VirB1-89K. Sequence alignment and phylogenetic analysis of VirB1-89K homologs were performed using MEGA 5.1 software. Values at nodes indicate bootstrap values for 500 replicates. (B) Analysis of the tertiary structure of the CHAP domain of Luminespib price VirB1-89K by using the online server SWISS-MODEL. (C) Visualization of the surface active site of the CHAP domain by using PyMOLviewer, showing Acadesine purchase the cysteine residue in green and histidine in red. Tertiary structure prediction showed that the CHAP domain of VirB1-89K belongs to the α + β structural class, with the N-terminal half containing 3 predicted α-helices and the

C-terminal half composed of 6 predicted β-strands (Figure 1B). Selleckchem SNS-032 protein tertiary structure modeling revealed that this CHAP domain Roflumilast contains an putative active center composed of a conserved cysteine and a histidine (Figure 1C), these two invariant residues form the main part of the active site of CHAP domain containing proteins [19, 21, 22]. These results together with the above phylogeny analysis

suggested that VirB1-89K may be an N-acetylmuramyl-L-alanine amidase. Expression and purification of the CHAP domain of VirB1-89K To figure out the function of VirB1-89K during the assembly of 89K T4SS apparatus, a 411 bp DNA fragment containing the CHAP domain of VirB1-89K was cloned and over-expressed in E. coli as a C-terminally His6-tagged protein. The protein of interest was designated VirB1-89KCHAP. We found VirB1-89KCHAP was efficiently expressed after induction at 16°C (Figure 2A). The molecular mass of the expressed recombinant protein agreed well with a predicted size of 15.4 kDa. Although a majority of the VirB1-89KCHAP protein was present in the inclusion body fractions of crude cell lysates, sufficient soluble material was produced to recover useful amounts of active protein. Highly purified protein (>95% homogeneity) was prepared by Ni+ affinity chromatography and gel filtration (Figure 2B). N-terminal sequencing results confirmed that the produced protein was indeed the CHAP domain of VirB1-89K. Figure 2 Over-expression and purification of VirB1-89KCHAP. (A) SDS-PAGE analysis (12%) of the interest VirB1-89KCHAP protein expressed in E. coli.