Biomedical subjects

W E Bentley

Publications and source records attributed to W E Bentley.

At least 19 recordsLinked to original sources

PubMed2001

Genomic analysis of high-cell-density recombinant Escherichia coli fermentation and "cell conditioning" for improved recombinant protein yield.

The Escherichia coli stress gene transcription profile and response to recombinant protein overexpression were substantially altered at high cell density when compared with low cell density. Reverse trascription-polymerase chain reaction RT-PCR-amplified mRNA from low (4 g[DCW]/L) and high-cell-density (43.5 g [DCW]/L) conditions were hybridized with a DNA microarray of Kohara clones encompassing 16% of the E. coli genome, and differentially displayed genes were identified. Transcript-specific RNA dot blots indicated that molecular chaperones (groEL, ibpA, degP), proteases (degP, ftsH), the lysis gene mltB, and DNA damage/bacteriophage-associated gene transcript levels (ftsH, recA, alpA, uvrB) increased 10- to 43-fold at high cell density. In addition, overexpression of recombinant green fluorescent protein (GFP(uv))/chloramphenicol acetyltransferase (CAT) fusion protein did not change the rates of cell growth or cell lysis. The stress gene transcription profile at high cell density was used to evaluate "cell conditioning" strategies to alter the levels of chaperones, proteases, and other intracellular proteins prior to recombinant protein overexpression. Interestingly, the addition of 1 g/L dithiothreitol (DTT) 20 min prior to induction of a GFP(uv)/CAT fusion protein resulted in a twofold increase in CAT activity when compared with the unconditioned controls. In addition, RNA dot blots of five stress genes confirmed that cell conditioning strategies significantly altered the dynamic stress gene response to foreign protein overexpression.

Bacterial Proteins↗

PubMed2001

Mapping stress-induced changes in autoinducer AI-2 production in chemostat-cultivated Escherichia coli K-12.

Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenic E. coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E. coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by E. coli for intercellular communication, was studied in E. coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-beta-D-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in E. coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.

Aerobiosis↗

PubMed2000

Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter.

Green fluorescent protein (GFP) was used to study the regulation of the galactose-inducible GAL1 promoter in yeast Saccharomyces cerevisiae strains. GFP was cloned into the pGAL110 vector and transformed into the yeast strains. Time course studies comparing culture fluorescence intensity and GFP concentration were conducted along with on-line monitoring of GFP expression. Our results demonstrated that GFP fluorescence could be used as a quantifiable on-line reporter gene in yeast strains. The effect of an integrated GAL10p-GAL4 transcription cassette was investigated. Induction time studies showed that there was no significant difference in GFP expression level by adding galactose at different culture times. A wide range of galactose concentrations was used to study the initial galactose concentration effect on GFP expression kinetics. A minimum of 0.05 g/L galactose doubled the GFP fluorescence signal as compared to the control, whereas 0.1 g/L gave the highest specific GFP yield. A simple analytical model was proposed to describe GFP expression kinetics based on the experimental results. In addition, this GFP-based approach was shown to have potential use for high-throughput studies. The use of GFP as a generic tool provided important insights to the GAL expression system and has great potential for further process optimization applications.

Blotting, Western↗

PubMed2000

Framework for online optimization of recombinant protein expression in high-cell-density Escherichia coli cultures using GFP-fusion monitoring.

A framework for the online optimization of protein induction using green fluorescent protein (GFP)-monitoring technology was developed for high-cell-density cultivation of Escherichia coli. A simple and unstructured mathematical model was developed that described well the dynamics of cloned chloramphenicol acetyltransferase (CAT) production in E. coli JM105 was developed. A sequential quadratic programming (SQP) optimization algorithm was used to estimate model parameter values and to solve optimal open-loop control problems for piecewise control of inducer feed rates that maximize productivity. The optimal inducer feeding profile for an arabinose induction system was different from that of an isopropyl-beta-D-thiogalactopyranoside (IPTG) induction system. Also, model-based online parameter estimation and online optimization algorithms were developed to determine optimal inducer feeding rates for eventual use of a feedback signal from a GFP fluorescence probe (direct product monitoring with 95-minute time delay). Because the numerical algorithms required minimal processing time, the potential for product-based and model-based online optimal control methodology can be realized.

Algorithms↗

PubMed2000

Evanescent wave long-period fiber bragg grating as an immobilized antibody biosensor.

An immunosensor using a long-period grating (LPG) was used for sensitive detection of antibody-antigen reactions. Goat anti-human IgG (antibody) was immobilized on the surface of the LPG, and detection of specific antibody-antigen binding was investigated. This sensor operates using total internal reflection where an evanescent field interacts with bound antibody immobilized over the grating region. The reaction between antibody and antigen altered the LPG transmission spectrum and was monitored in real time as a change in refractive index, thereby eliminating the need for labeling antigen molecules. Human IgG binding was observed to be concentration dependent over a range of 2-100 microg mL-1, and equilibrium bound antigen levels could be attained in approximately 5 min using an initial rate determination. Binding specificity was confirmed using human interleukin-2 and bovine serum albumin as controls, and nonspecific adsorption of proteins did not significantly interfere with detection of binding. Antigen detection in a heterogeneous protein mixture and in crude cell lysate from Escherichia coli was also confirmed. Moreover, regeneration of the LPG surface via diethylamine treatment resulted in approximately 80% removal of bound antigen. Subsequently, fibers reexposed to antigen retained greater than 85% of the initial signal after five consecutive regeneration cycles.

Animals↗

PubMed2000

Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility.

We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion. The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies.

Chloramphenicol O-Acetyltransferase↗

PubMed2000

A comparative study of global stress gene regulation in response to overexpression of recombinant proteins in Escherichia coli.

Global gene regulation throughout the Escherichia coli stress response to overexpression of each of five recombinant proteins was evaluated. Reverse-transcriptase polymerase chain reaction-amplified mRNA from induced and control cells were hybridized with a DNA array of Kohara clones representing 16% (700 genes) of the E. coli genome. Subsequently, Northern analysis was performed for quantification of specific gene dynamics and statistically significant overlap in the regulation of 11 stress-related genes was found using correlation analysis. The results reported here establish that there are dramatic changes in the transcription rates of a broad range of stress genes (representing multiple regulons) after induction of recombinant protein. Specifically, the responses included significantly increased upregulation of heat shock (ftsH, clpP, lon, ompT, degP, groEL, aceA, ibpA), SOS/DNA damage (recA, lon, IS5 transposase), stationary phase (rpoS, aceA), and bacteriophage life cycle (ftsH, recA) genes. Importantly, similarities at the microscopic (gene) level were not clearly reflected at the macroscopic (growth rate, lysis) level. The use of such dynamic data is critical to the design of gene-based sensors, the engineering of metabolic pathways, and the determination of parameters (harvest and induction times) needed for successful recombinant E. coli fermentations.

Bacteriophage lambda↗

PubMed2000

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase.

Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5' end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.

Aryldialkylphosphatase↗

PubMed2000

Antisense downregulation of sigma(32) as a transient metabolic controller in Escherichia coli: effects on yield of active organophosphorus hydrolase.

Plasmids containing an antisense fragment of the sigma(32) gene were constructed and introduced into Escherichia coli cells. Downregulation of the sigma(32)-mediated stress response was evaluated under heat shock and ethanol stress and during the production of organophosphorus hydrolase (OPH). Northern blot analyses revealed that sigma(32) sense mRNA was virtually undetected in antisense-producing cultures from 5 to 20 min after antisense induction. However, lower-molecular-weight bands were found, presumably due to partial degradation of sigma(32) mRNA. While a >10-fold increase in sigma(32) protein level was found under ethanol stress in the control cultures, antisense producing cultures resulted in a <3-fold increase, indicating downregulation of sigma(32). Correspondingly, antisense synthesis resulted in a decreased level of a sigma(32) regulated chaperone (GroEL) for the first 2 h after induction relative to control cultures without sigma(32) antisense mRNA. The total yield of OPH in the presence of sigma(32) antisense was, on average, 62% of the yield without antisense. However, during sigma(32) antisense production, a sixfold-higher specific OPH activity was observed compared to non-antisense-producing cultures.

Aryldialkylphosphatase↗

PubMed2000

OmpT expression and activity increase in response to recombinant chloramphenicol acetyltransferase overexpression and heat shock in E. coli.

The activity of a 35 kDa protease increased in response to induced expression of chloramphenicol acetyltransferase (CAT) in E. coli. This protease was partially purified, extensively characterized, and identified via the use of zymogram gels as the outer membrane protease, OmpT. In experiments targeting the overlap of well-characterized stress responses, OmpT activity was found to increase in response to heat shock but was only minimally affected by amino acid limitation. The largest increase in activity was found after induction of CAT. OmpT expression levels also increased in response to induction of recombinant CAT overexpression and heat shock. This is the first report of increased activity and expression of an outer membrane protease during cytoplasmic overexpression of a recombinant protein.

Bacterial Outer Membrane Proteins↗

PubMed1999

Insect larval expression process is optimized by generating fusions with green fluorescent protein.

The insect larvae/baculovirus protein production process was dramatically simplified by expressing fusion proteins containing green fluorescent protein (GFP) and the product-of-interest. In this case, human interleukin-2 (hIL-2) and chloramphenicol acetyl-transferase (CAT) were model products. Specifically, our fusion construct was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), the UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of the product from the fusion, and the product, hIL-2 or CAT. Both the approximately 52 kDa GFPuv/hIL-2 and approximately 63 kDa GFPuv/CAT fusions were expressed in Trichoplusia ni larvae at 9.0 microg-hIL-2 and 24.1 microg-CAT per larva, respectively. The GFP enabled clear identification of the infection process, harvest time, and more importantly, the quantity of product protein. Because the GFP served as a marker, this technique obviates the need for in-process Western analyses (during expression, separation, and purification stages). As a purification marker, GFP facilitated rapid identification of product-containing elution fractions (Cha et al., 1999b), as well as product-containing waste fractions (e.g., cell pellet). Also, because the fluorescence intensity was linear with hIL-2 and CAT, we were able to select the highest-producing larvae. That is, three fold more product was found in the brightest larva compared to the average. Finally, because the GFP is attached to the product protein and the producing larvae can be selected, the infection and production processes can be made semi-continuous or continuous, replacing the current batch process. These advantages should help to enable commercialization of larvae as expression hosts.

Animals↗

PubMed1999

Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor.

Synthesis of an operon fusion protein was investigated in batch and fed-batch cultures at high cell densities of recombinant Escherichia coli JM105 [pBAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L(-1) DCW) to be attained. This was believed to be the highest reported value for dry cell mass of E. coli strain JM105 expressing two recombinant proteins. Transcription of the two reporter genes, green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT), was under the control of the p(BAD) promoter of the araBAD (arabinose) operon. Each protein was independently translated via separate ribosome binding sites. CAT served as a model recombinant protein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monitored on-line using an intensity-based optical sensor. A linear correlation between the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product under conditions of high biomass concentration and high productivity.

Arabinose↗

PubMed1999

Chimeric infectious bursal disease virus-like particles expressed in insect cells and purified by immobilized metal affinity chromatography.

Chimeric virus-like particles (VLPs) of infectious bursal disease virus (IBDV) were produced by coinfecting Spodoptera frugiperda (Sf-9) insect cells with two recombinant baculoviruses, vIBD-7 and vEDLH-22. vIBD-7 encodes VP2, VP3, and VP4 of the IBDV structural proteins. vEDLH-22 encodes VP2 with five histidine residues at the carboxy-terminus (VP2H). Coinfection produced hybrid VLPs composed of VP2, VP2H, and VP3. The additional histidine residues on VP2H enabled the efficient purification of VLPs based on immobilized metal affinity chromatography (IMAC). These results demonstrated that the VLPs formed are comprised of chimeric subunits with attached affinity ligands, and further, that sufficient His5 ligand was available for binding to the IMAC metal-chelating resin. Additionally, these novel particles were fully characterized for antigenicity by a series of monoclonal antibodies, and appeared identical to the two wild-type IBDV strains contributing subunits to the chimeric VLP. IMAC purification provides a promising low-cost and simple scheme to purify VLPs as vaccines.

Animals↗

PubMed1999

Characterization of stress and protein turnover from protein overexpression in fed-batch E. coli cultures.

A structured kinetic model that accounts for proteolytic degradation due to recombinant protein overexpression is introduced and its performance evaluated by comparison with previously reported fed-batch experimental data. This mathematical model contains an additional pool for a generic key precursor (in our case phenylalanine), an improved IPTG transport term, a phenylalanine transport term, and a variable protein turnover expression that accounts for proteolytic activity. The model predictions concerning proteolytic activity, glucose level, and cell growth are in very good agreement with an amino acid depletion hypothesis. Cultures exposed to greater stress showed higher and/or longer proteolysis, whereas less overall proteolytic activity was observed when the effect of induction was somewhat ameliorated.

Chloramphenicol O-Acetyltransferase↗

PubMed1999

Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells.

A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system.

Animals↗

PubMed1999

Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects and partial control of proteolysis.

Many eukaryotic proteins have been successfully expressed in insect cells infected with a baculovirus in which the foreign gene has been placed under the control of a viral promoter. This system can be costly at large scale due to the quality of virus stock, problems of oxygen transfer, and severity of large-scale contamination. To circumvent this problem, we have investigated the expression of a foreign protein, human interleukin-2 (IL-2), in insect larvae, Trichoplusia ni, infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The IL-2 gene was placed under control of the p10 promoter so that the polyhedra remained intact for efficient primary infection. From our results, it was clear that early infection limited larval growth and late infection delayed product production until near pupation, hence infection timing was important. Also, the harvest time was crucial for obtaining high yield, because IL-2 production had a sharp optimal peak with a time of occurrence dependent on both temperature and the initial amount of infection virus. Specifically, we found that, by raising the infection temperature to 30 degrees C, we more than doubled the protein productivity. Furthermore, a significant concern of the larvae/baculovirus expression system has been the large amount of protease produced by the larvae, which adversely affects the protein yield. Therefore, we screened several protease inhibitors and characterized the larval protease specificity and timing to attenuate their impact. This report elucidates and delineates the factors that most directly impact protein yield in the larvae expression system, using IL-2 as a model.

Animals↗

PubMed1999

Reverse transcription-PCR differential display analysis of Escherichia coli global gene regulation in response to heat shock.

A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in Escherichia coli. A novel combination of primers designed specifically for the start and stop regions of E. coli genes (based on the findings of Fislage et al. [R. Fislage, M. Berceanu, Y. Humboldt, M. Wendt, and H. Oberender, Nucleic Acids Res. 25:1830-1835, 1997]) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR. The validity of the technique was demonstrated by applying it to heat shock analysis. Specifically, RT-PCR-amplified total RNA from heat-shocked and non-heat-shocked cells were hybridized with slot blots of the Kohara set (U. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987; S. Chuang, D. Daniels, and F. Blattner, J. Bacteriol. 175:2026-2036, 1993). The signals obtained for heat-shocked and control cultures of each clone were compared, and differences in intensity were evaluated by calculating induction ratios. Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation. Also, for several genes, Northern blotting and total RNA dot blotting were performed to confirm that the transcript levels in the original RNA samples were different. This technique extended previously described methods for studying global gene regulation in E. coli by incorporating a PCR amplification step in which global, mRNA-specific primers were used. In addition, the method employed here can be easily extended to study E. coli global gene regulation in response to additional environmental stimuli.

Base Sequence↗

PubMed1999

Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells.

We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate Escherichia coli cellular stress responses in quiescent or resting cells. Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor sigma32, the protease subunit ClpB, and the chaperone DnaK, to the reporter gene gfpuv. When perturbed by a chemical or physical stress (such as a heat shock, nutrient [amino acid] limitation, or addition of IPTG [isopropyl-beta-D-thiogalactopyranoside], acetic acid, ethanol, phenol, antifoam, or salt [osmotic shock]), the E. coli cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence. Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded.

Blotting, Western↗