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Biomedical subjects

W E Bentley

Publications and source records attributed to W E Bentley.

At least 19 recordsLinked to original sources
PubMed2003

Biotechnological applications of green fluorescent protein.

Since its first use as a reporter gene in 1994, green fluorescent protein (GFP) has served as the researcher's agent: slipping, virtually undetected, into unseen spaces, reporting back valuable information, and securing the delivery of precious cargo through hostile domains. GFP's strength lies in its small size, formidable stability, and relative ease of use. It requires only oxygen and an energy source to do its work, which can be supplied at low cost and high precision, respectively. With such a low threshold for use, GFP is often the first line of inquiry into an unknown space. Here is provided a brief compendium of GFP's contributions to biotechnology. They are linked by a need for a level of information that was previously inaccessible, both spatially and temporally. Protein fusions, transcriptional reporters, whole-organism visualization, and various other screening applications are reviewed with respect to biotechnological applications. Germane molecular improvements to GFP are also discussed.

Animals↗
PubMed2001

Quorum signaling via AI-2 communicates the "Metabolic Burden" associated with heterologous protein production in Escherichia coli.

Recent reports have shown that bacterial cell-cell communication or quorum sensing is quite prevalent in pathogenic Escherichia coli, especially at high cell density; however, 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 in overexpression of plasmid-encoded genes. In this work, it was found that the activity of a quorum signaling molecule, autoinducer-2 (AI-2), decreased significantly following induction of several plasmid-encoded genes in both low and high-cell-density cultures of E. coli. Furthermore, we show that AI-2 signaling level was linearly related to the accumulation level of each protein product and that, in general, the highest rates of recombinant protein accumulation resulted in the greatest attenuation of AI-2 signaling. Importantly, our findings demonstrate for the first time that recombinant E. coli communicate the stress or burden of overexpressing heterologous genes through the quorum-based AI-2 signaling pathway.

Bacterial Proteins↗
PubMed2001

Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions.

It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000). In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)). Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH). Importantly, the fusion protein was still fluorescent as determined via microscopy. In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone.

Artificial Gene Fusion↗
PubMed2001

Effect of MOI ratio on the composition and yield of chimeric infectious bursal disease virus-like particles by baculovirus co-infection: deterministic predictions and experimental results.

Virus-like particles (VLPs) are empty particles consisting of virus capsid proteins that closely resemble native virus but are devoid of the native viral nucleic acids and therefore have attracted significant attention as noninfectious vaccines. A recombinant baculovirus, vIBD-7, which encodes the structural proteins (VP2, VP3, and VP4) of infectious bursal disease virus (IBDV), produces native IBD VLPs in infected Spodoptera frugiperda insect cells. Another baculovirus, vEDLH-22, encodes VP2 that is fused with a histidine affinity-tag (VP2H) at the C-terminus. By co-infection with these two baculoviruses, hybrid VLPs with histidine tags were formed and purified by immobilized metal affinity chromatography (Hu et al., 1999). Also, we demonstrated that varying the MOI ratio of these infecting viruses altered the extent of VP2H incorporated into the particles. A dynamic mathematical model that described baculovirus infection and VLP synthesis (Hu and Bentley, 2000) was adapted here for co-infection and validated by immunofluorescence labeling. It was shown to predict the VLP composition as a dynamic function of MOI. A constraint in the VP2H content incorporated into the particles was predicted and shown by experiments. Also, the MOI ratio of both infecting viruses was shown to be the major factor influencing the composition of the hybrid particles and an important factor in determining the overall yield. ELISA results confirmed that VP2H was exhibited to a varied extent on the outer surface of the particles. This model provides insight on the use of virus co-infection in virus-mediated recombinant protein expression systems and aids in the optimization of chimeric VLP synthesis.

Animals↗
PubMed2001

Stochastic kinetic analysis of the Escherichia coli stress circuit using sigma(32)-targeted antisense.

A stochastic Petri net model was developed for simulating the sigma(32) stress circuit in E. coli. Transcription factor sigma(32) is the principal regulator of the response of E. coli to heat shock. Stochastic Petri net (SPN) models are well suited for kinetics characterization of fluxes in biochemical pathways. Notably, there exists a one-to-one mapping of model tokens and places to molecules of particular species. Our model was validated against experiments in which ethanol (inducer of heat shock response) and sigma(32)-targeted antisense (downward regulator) were used to perturb the sigma(32) regulatory pathway. The model was also extended to simulate the effects of recombinant protein production. Results show that the stress response depends heavily on the partitioning of sigma(32) within the cell; that is, sigma(32) becomes immediately available to mediate a stress response because it exists primarily in a sequestered, inactive form, complexed with chaperones DnaK, DnaJ, and GrpE. Recombinant proteins, however, also compete for chaperone proteins, particularly when folded improperly. Our simulations indicate that when the expression of recombinant protein has a low requirement for DnaK, DnaJ, and GrpE, the overall sigma(32) levels may drop, but the level of heat shock proteins will increase. Conversely, when the overexpressed recombinant protein has a strong requirement for the chaperones, a severe response is predicted. Interestingly, both cases were observed experimentally.

Antisense Elements (Genetics)↗
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

Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay.

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.

Animals↗
PubMed2001

Combinatorial screening for enzyme-mediated coupling. Tyrosinase-catalyzed coupling to create protein--chitosan conjugates.

In nature, tyrosinase-generated o-quinones are commonly involved in processes that lead to functional biomaterials. These biomaterials are chemically complex and have been difficult to analyze. Furthermore, the cascade of reactions involving o-quinones is poorly understood, and it has been difficult to mimic ex vivo for materials processing. We report the use of a combinatorial approach to learn how tyrosinase and low molecular weight phenolic precursors can be used to generate biologically active protein-polysaccharide conjugates. Specifically, we screened various phenolic coupling precursors and various reaction conditions for the coupling of proteins onto the polysaccharide chitosan. Several natural phenols were identified as appropriate precursors for the coupling of polyhistidine tagged organophosphorus hydrolase (His-OPH) onto chitosan films. OPH activity was retained upon coupling and subsequent studies indicated that the histidine tag was not necessary for coupling. Using conditions identified for His-OPH coupling, we observed that various biologically active proteins (cytochrome c, OPH, and His-CAT) could be coupled onto chitosan films. The glycosylated protein horseradish peroxidase was not effectively coupled onto chitosan under the conditions studied. In all cases studied, we observed that coupling required a phenolic precursor, suggesting that tyrosinase is unable to couple by reaction with surface tyrosyl residues of the target protein. In conclusion, this study illustrates a combinatorial approach for the "discovery" of conditions to couple biologically active proteins onto chitosan through natural, quinone-based processes.

Biocompatible Materials↗
PubMed2001

DNA microarray-based identification of genes controlled by autoinducer 2-stimulated quorum sensing in Escherichia coli.

Bacterial cell-to-cell communication facilitates coordinated expression of specific genes in a growth rate-II and cell density-dependent manner, a process known as quorum sensing. While the discovery of a diffusible Escherichia coli signaling pheromone, termed autoinducer 2 (AI-2), has been made along with several quorum sensing genes, the overall number and coordination of genes controlled by quorum sensing through the AI-2 signal has not been studied systematically. We investigated global changes in mRNA abundance elicited by the AI-2 signaling molecule through the use of a luxS mutant that was unable to synthesize AI-2. Remarkably, 242 genes, comprising ca. 5.6% of the E. coli genome, exhibited significant transcriptional changes (either induction or repression) in response to a 300-fold AI-2 signaling differential, with many of the identified genes displaying high induction levels (more than fivefold). Significant induction of ygeV, a putative sigma(54)-dependent transcriptional activator, and yhbH, a sigma(54) modulating protein, suggests sigma(54) may be involved in E. coli quorum sensing.

Amino Acid Sequence↗
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↗
PubMed2001

High throughput studies of gene expression using green fluorescent protein-oxidative stress promoter probe constructs: the potential for living chips.

Green fluorescent protein fusions were constructed with several oxidative stress promoters from Escherichia coli. These promoters were chosen for their induction by reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals. When exposed to various free radical insults, the cells fluoresced with great specificity based on the corresponding ROS. In this work, we propose a way in which these constructs could be used to study the mode of action of a variety of antitumor drugs. This approach offers the possibility of complementing gene chip technology by the creation of living chips for high throughput screening as well as studying differential gene expression.

Antineoplastic Agents↗
PubMed2001

Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors.

In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5-30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.

Journal Article↗
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↗