Optimal Expression Conditions Research Articles

Biochemical Characterization of VlmL, a Seryl-tRNA Synthetase Encoded by the Valanimycin Biosynthetic Gene Cluster

Previous studies have shown that the valanimycin producer Streptomyces viridifaciens contains two genes encoding proteins that are similar to seryl-tRNA synthetases (SerRSs). One of these proteins (SvsR) is presumed to function in protein biosynthesis, because it exhibits a high degree of similarity to the single SerRS of Streptomyces coelicolor. The second protein (VlmL), which exhibits a low similarity to the S. coelicolor SerRS, is hypothesized to play a role in valanimycin biosynthesis, because the vlmL gene resides within the valanimycin biosynthetic gene cluster. To investigate the role of VlmL in valanimycin biosynthesis, VlmL and SvsR have been overproduced in soluble form in Escherichia coli, and the biochemical properties of both proteins have been analyzed and compared. Both proteins were found to catalyze a serine-dependent exchange of 32P-labeled pyrophosphate into ATP and to aminoacylate total E. coli tRNA with L-serine. Kinetic parameters for the two enzymes show that SvsR is catalytically more efficient than VlmL. The results of these experiments suggest that the role of VlmL in valanimycin biosynthesis is to produce seryl-tRNA, which is then utilized for a subsequent step in the biosynthetic pathway. Orthologs of VlmL were identified in two other actinomycetes species that also contain orthologs of the S. coelicolor SerRS. The significance of these findings is herein discussed.

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3 h in PBG (PBS containing 0.5% BSA and 5 mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5 h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01 μg/ml or greater, and by zymosan concentrations of 0.1 μg/ml or greater. ROS production in response to incubation with PMA was signficantly increased starting at 10 −7 M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10 −7 M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3 h in RPMI with LPS (0.1 μg/ml), zymosan (10 μg/ml), PGN (10 μg/ml), and PMA (10 −7 M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.

North and South American Loxosceles spiders: Development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab′) 2 preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti- Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.

Enhancing functional production of G protein‐coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.

Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression

Soybean mosaic virus (SMV)-CN18 is an Rsv resistance-breaking (RB) isolate to overcome soybean resistance genes Rsv1, Rsv3 and Rsv4. The aim of this study was to characterize nuclear inclusion protein a (NIa protein) of RB isolate at the molecular level and demonstrate its processing into genome-linked protein (VPg) and NIa-Pro domains in Esherichia coli containing a bacterial expression pET vector inserted with NIa gene. The full-length of NIa gene was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) and its 1298 nucleotides (nt) and 432 amino acids (aa) were deduced. The nt and aa sequences of NIa gene of SMV-CN18 shared high identities with the corresponding sequences of the NIa gene of the known SMV isolates, suggesting that the NIa is a highly conserved protein. The NIa-Pro domain contains a highly conserved structural motif for proteolysis, while the VPg domain contains a nuclear localization signal (NLS), a putative NTP-binding site and cellular factor-binding sites. The phylogenetic tree revealed that less divergence of NIa protein exists among twelve SMV isolates, which can be supported by a low bootstrap value between clades. In addition, the full-length of NIa gene, amplified by RT-PCR, was ligated into pET-28b E. coli expression vector with an N-terminal His6-tag. Optimal conditions for expression were at 1mM treatment of IPTG at 25°C for 5 hr. The released protein from bacterial lysates remained soluble and proved the processing form of the NIa polyprotein. E. coli expression system shows the processed product of 29 kDa VPg in SDS-PAGE confirmed by western blot analysis in both crude extracts and purified elution products, using Ni2+-NTA resin. The present study indicates that the N-terminal region of NIa which is processed and expressed in bacteria.

The production of cleaved, trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein vaccine antigens and infectious pseudoviruses using linear polyethylenimine as a transfection reagent

Trimeric HIV-1 envelope glycoproteins (Env) are now being evaluated instead of monomeric gp120 as vaccine antigens because they mimic more closely the spikes expressed on the surface of virions. Thus, it can be argued that trimers have a more native structure than gp120, so might be superior at raising neutralizing antibodies. One approach to making Env trimers is to ensure that they are cleaved at the gp120–gp41 border, but stabilized by other, engineered substitutions such as intra-subunit disulfide bonds (SOS and SOSIP gp140 proteins). However, the production of properly folded, cleaved trimers is complicated by the requirement for co-expression of the exogenous protease furin, to facilitate the efficient processing oft the gp120–gp41 cleavage site. Also, yields of purified trimeric SOSIP gp140 proteins are usually moderate and for scale-up procedures the cost of transfection reagents becomes an important economical factor. Here, we assess the optimal culture conditions for the transient expression of these complex proteins. We found that the use of linear polyethylenimine 25 kDa (PEI25k) as a transfection aid was a cost-efficient, economical alternative to several commercially available products. By using PEI25k and an optimized plasmid:furin ratio, we could express proteolytically mature, trimeric Env vaccine antigens at levels high enough for use in immunization or structural studies. We also show that the same transfection method can be used to generate infectious pseudoviruses.

High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells

To develop a highly efficacious method for preparation of soluble SARS S-protein using adenovirus vector to meet the requirement for S-protein investigation. The human adenovirus vector was used to express the soluble S-protein (corresponding to 1 approximately 1190 amino acids) fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells. Under the conditions of infection dose (MOI of 50) and expression time (48 h), the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 microg/10(6) cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells,demonstrating that the soluble S-protein could remain the biologic activity in the native molecule. The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.

Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors

BackgroundThe solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC).ResultsA single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods.ConclusionThe experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression.

Optimization of expression of dhaT gene encoding 1,3‐propanediol oxidoreductase from Klebsiella pneumoniae in Escherichia coli using the methods of uniform design and regression analysis

AbstractThis paper is concerned with the optimization of expression conditions of 1,3‐propanediol oxidoreductase (PDOR) using the uniform design (UD) method and regression analysis. The concentration of the inducer isopropyl‐β‐D‐thiogalactopyranoside (IPTG), the induction temperature and the induction time, which were identified as three crucial factors, were optimized in 50 mL cultivation tubes containing 5 mL LB culture. The optimal expression conditions for enhanced PDOR activity were found to be an IPTG concentration of 0.1 mmol l−1, an induction temperature of 21.2 °C and an induction time of 5 h. Subsequent experimental trials confirmed the validity of the model. These optimal expression conditions in the cultivation tube culture led to a PDOR activity of 9.25 U mL−1, which was considerably higher than those obtained in the optimizing experimental trials. Copyright © 2005 Society of Chemical Industry

Soluble expression, purification, and stabilization of a pro-apoptotic human protein, CARP

CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was purified to near homogeneity by a two-step FPLC procedure, first by Ni 2+ affinity chromatography followed by a gel-filtration chromatography, which yielded about 10 mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-filtration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2 mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulfide bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulfide bonds and to stabilize the protein. Successfully growing crystals of the purified CARP protein also proved that we can produce well folded CARP protein in E. coli.

Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells

Rapid expression of recombinant proteins for structure determination is one of the major challenges in pharmaceutical and academic research, since the number of potential drug targets has increased significantly in the last decade. Despite the fact that the baculovirus expression vector system is widely used for this purpose, the system is hampered by three very slow and tedious procedures, namely generation of high titer baculovirus stock, determination of the virus titer and discovery of the best conditions for protein expression. We herein describe the development of the ultraBac system to address and overcome these issues for protein expression in insect cells. We have established a new baculovirus expression technology for insect cells that is based on co-expression of GFP with target genes, a new regime for cell culturing and a highly efficient purification and enrichment procedure for recombinant baculovirus particles. Co-expression of GFP is used to monitor the infection of insect cells, to simplify titer determination and to optimize expression conditions. The new regime for cell culturing with increased viability of non-infected insect cells and its combination with the massive enrichment of virus particles via high-speed centrifugation enables the production of large amounts of recombinant virus in a very short period of time. By combining these techniques and by using the bicistronic vector pUltraBac-1, we have been able to cut the time-lines for protein expression in insect cells by half, approaching those for protein production in Escherichia coli. This new expression system is a significant step forward towards industrialized protein production in both, industry and academia.

Conditions affecting hydrodynamics‐based gene delivery into mouse liver in vivo

1. It has been demonstrated that the hydrodynamics-based procedure has high efficiency to deliver foreign genes into the liver. The widespread use of this procedure in gene function studies and as a treatment option for liver and other organ diseases puts considerable importance on the investigation of various conditions that affect hydrodynamics-based gene delivery into mouse liver in vivo. 2. Various conditions, including the volume, speed and solution of the injection and the state, gender and strain of the animal were manipulated to evaluate their effect on the expression levels in mice of human factor IX (hFIX) 8 h after tail vein injection of the plasmid pCMV-hFIX. 3. It was found that an injection volume of 2-2.5 mL and an injection speed of 5-7 s were very effective in delivering DNA into the mouse liver. Using Ringer's solution as an injection fluid increased the efficiency of hFIX expression. 4. Anaesthetized mice expressed higher hFIX than conscious mice. Males expressed higher hFIX than females. The ICR mouse strain demonstrated higher expression of the foreign gene than did the C57 strain. 5. The effects of these specific factors on hFIX expression may be caused by variations in hydrostatic pressure, the degree of liver damage and liver size. 6. It can be concluded that there are optimal conditions for hFIX expression in the liver. This information may be helpful for the application of hydrodynamics-based procedures in the investigation of gene expression and gene therapy.

Expression and characterization of Gag protein of HIV-1 CN in Pichia pastoris

To express the core protein of HIV-1 of Chinese prevalent strain (HIV-1 CN) in Pichia pastoris, the full-length gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by SalI was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA (1.3 kb) was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55 kDa, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13% of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows: BMMY medium, 80–90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85%. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.

Expression of the melittin gene of Apis cerana cerana in Escherichia coli

A cDNA encoding melittin in Apis cerana cerana was obtained by PCR from the recombinant plasmid and cloned into the GST fusion expression vector pGEX-4T-2 for expression of the protein. The expressed protein of about 29 kDa was detected by Western blot and triple antibody sandwich ELISA, indicating that the recombinant protein is the fusion protein of GST-AccM. The expression conditions of GST-AccM fusion protein for Escherichia coli BL21 transformant were optimized. Thin layer scanning on the SDS–PAGE profiles of GST-AccM showed that the expressed protein accumulated up to about 15.2% of total protein of bacterial cells under the optimized expression condition. Purified and recovered recombinant melittin of A. cerana cerana showed bioactivity in activating rabbit platelets to aggregate.

Inhibition of Nitric-oxide Synthase-I (NOS-I)-dependent Nitric Oxide Production by Lipopolysaccharide plus Interferon-γ Is Mediated by Arachidonic Acid: EFFECTS ON NFκB ACTIVATION AND LATE INDUCIBLE NOS EXPRESSION

Previous results have indicated that lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) inhibits nitric-oxide synthase (NOS)-I activity in glial cells. We report here that arachidonic acid (AA) plays a pivotal role in this response, which was consistently reproduced in different glial cell lines and in primary rat astrocytes. This notion was established using pharmacological inhibitors of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2) antisense oligonucleotides, and AA add-back experiments. This approach not only allowed the demonstration that AA promotes inhibition of NOS-I activity but also produced novel experimental evidence that LPS/IFNgamma itself is a potential stimulus for NOS-I. Indeed, LPS/IFNgamma fails to generate nitric oxide (NO) via NOS-I activation simply because it activates the AA-dependent signal that impedes NOS-I activity. Otherwise, LPS/IFNgamma promotes NO formation, sensitive to exogenous AA, in cells in which cPLA2 is pharmacologically inhibited or genetically depleted. Because NO suppresses the NFkappaB-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFNgamma-induced AA pathway provides optimal conditions for NFkappaB activation and subsequent NOS-II expression. Inhibition of cPLA2 activity, while reducing the availability of AA, consistently inhibited NFkappaB activation and NOS-II mRNA induction and delayed NO formation. These responses were promptly reestablished by addition of exogenous AA. Finally, we have demonstrated that the LPS/IFNgamma-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.

Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies

Phospholamban (PLB) and Sarcolipin (SLN) are integral membrane proteins that regulate muscle contractility via direct interaction with the Ca-ATPase in cardiac and skeletal muscle, respectively. The molecular details of these protein–protein interactions are as yet undetermined. Solution and solid-state NMR spectroscopies have proven to be effective tools for deciphering such regulatory mechanisms to a high degree of resolution; however, large quantities of pure recombinant protein are required for these studies. Thus, recombinant PLB and SLN production in Escherichia coli was optimized for use in NMR experiments. Fusions of PLB and SLN to maltose binding protein (MBP) were constructed and optimal conditions for protein expression and purification were screened. This facilitated the large-scale production of highly pure protein. To confirm their functionality, the biological activities of recombinant PLB and SLN were compared to those of their synthetic counterparts. The regulation of Ca-ATPase activity by recombinant PLB and SLN was indistinguishable from the regulation by synthetic proteins, demonstrating the functional integrity of the recombinant constructs and ensuring the biological relevance of our future structural studies. Finally, NMR spectroscopic conditions were established and optimized for use in investigations of the mechanism of Ca-ATPase regulation by PLB and SLN.

Adaptation of a sandwich enzyme-linked immunosorbent assay to determine the concentration of bovine leukemia virus p24 and optimal conditions for p24 expression in short-term cultures of peripheral blood mononuclear cells

Bovine leukemia virus (BLV) is a common retroviral infection of cattle. Infection is accompanied by integration of BLV into the host cell genome and is persistent for the life of the individual as is the presence of anti-BLV antibodies. Lymphosarcoma occurs in a small fraction of infected adult individuals but otherwise there is little or no associated disease. Viremia is undetectable, however, BLV is expressed readily once infected cells are cultured in vitro. A sandwich enzyme-linked immunosorbent assay (sELISA) was optimized, using murine monoclonal antibodies, to quantify the major internal structural protein (p24) produced in short-term cultures of peripheral blood mononuclear cells (PBMCs). Optimal production of BLV p24 was achieved utilizing RPMI supplemented with 10% fetal bovine serum (FBS), pH 7, and 5×10 6 cells per ml. Cultures were terminated at 24 h. The sELISA was linear between 30 and 900 ng/ml and the limit of detection was 1.2 ng/ml. At three concentrations of p24, intra- and inter-assay coefficients of variation (CV) varied between 9.2 and 13.3 and 5.1 and 12.9%, respectively.

A Rubredoxin based system for screening of protein expression conditions and on-line monitoring of the purification process

Rubredoxin (Rub) from Thermotoga maritima, a 6.1-kDa red protein containing an Fe(III)-cysteine 4 center, was evaluated for its usefulness as a colored fusion tag for expression of recombinant proteins in E. coli. Here, we describe the Rub features relevant to accelerating screening for optimal high yield soluble expression conditions and automating the ensuing purification process. Spectroscopic properties and the yield of Rub fused to a typical target protein were compared to analogous GFP and Flavodoxin constructs, showing Rub absorption to be sufficient for structural genomics purposes while being produced at much higher soluble levels than GFP constructs. Based entirely on Rub absorption at 380 nm, both generic and affinity purification of crude cell lysate were performed: thus guided anion exchange purification of a Rub fusion construct as well as automated Ni-NTA purification resulted in pure protein. Rub is stable over a wide range of pH, temperature, and buffer environments, enabling robust purification protocols. Across a variety of fusion constructs, including N- and C-terminal Rub, quantitation via the Rub signal was shown to reliably correlate with analytical HPLC data obtained at 220 nm. We propose the “RubyTag” as an alternative to conventional protein fusion tags, as it combines a specific absorption signal with convenient biochemical and biological properties. Further, it allows direct on-line readout on conventional chromatography systems, holding promise for automated multi-step chromatography.

ToxR regulon of Vibrio cholerae and its expression in vibrios shed by cholera patients.

Toxigenic Vibrio cholerae cause cholera, a severe diarrheal disease responsible for significant morbidity and mortality worldwide. Two determinants, cholera enterotoxin (CT) and toxin coregulated pilus (TCP) are critical factors responsible for this organism's virulence. The genes for these virulence determinants belong to a network of genes (the ToxR regulon) whose expression is modulated by transcriptional regulators encoded by the toxRS, tcpPH, and toxT genes. To define the ToxR regulon more fully, mutants defective in these regulatory genes were transcriptionally profiled by using V. cholerae genomic microarrays. This study identified 13 genes that were transcriptionally repressed by the toxT mutation (all involved in CT and TCP biogenesis), and 27 and 60 genes that were transcriptionally repressed by the tcpPH and toxRS mutations, respectively. During the course of this analysis, we validated the use of a genomic DNA-based reference sample as a means to standardize and normalize data obtained in different microarray experiments. This method allowed the accurate transcriptional profiling of V. cholerae cells present in stools from cholera patients and the comparison of these profiles to those of wild-type and mutant strains of V. cholerae grown under optimal conditions for CT and TCP expression. Our results suggest that vibrios present in cholera stools carry transcripts for these two virulence determinants, albeit at relatively low levels compared with optimal in vitro conditions. The transcriptional profile of vibrios present in cholera stools also suggests that the bacteria experienced conditions of anaerobiosis, iron limitation, and nutrient deprivation within the human gastrointestinal tract.

High-level Expression of the ORF6 Gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Pichia pastoris

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5′-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZαA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23 kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0 g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.