Purification of recombinant green fluorescent protein using chromatofocusing with a pH gradient composed of multiple stepwise fronts.

Green fluorescent protein (GFP), which fluoresces in the green region of the visible spectrum and is widely used as a reporter for gene expression and regulation, was overexpressed in the JM105 strain of Escherichia coli transformed with pBAD-GFP. A two-step chromatofocusing procedure was used to purify GFP starting from cell lysate, with each step employing a pH gradient extending from pH 5.5 to 4.0. The first chromatofocusing step was performed using a low-pressure column in which a retained stepwise pH front formed by adsorbed buffering species was used to capture GFP directly from clarified cell lysate and selectively focus it into a chromatographic band. The second step utilized a high-performance column under mass overloaded conditions where a similar pH front acted as a protein displacer and led to the formation of a highly concentrated rectangular band of GFP. The overall procedure yielded a 50-fold increase in purity, a 20-fold volume reduction, and a recovery and purity for GFP of 60% and 80%, respectively. Because the method employs a strong-base ion-exchange column packing and low-cost buffers formed with formic and acetic acids instead of the proprietary column packings and polyampholyte elution buffers more generally used for chromatofocusing, it appears to be a practical alternative for the preparative ion-exchange chromatography of GFP in particular and for the recovery of recombinant proteins from cell lysate in general. A discussion is also given concerning the choice of appropriate buffers for the rational design of pH gradients involving retained, stepwise pH fronts that span a given pH range and of the use of the fluorescence properties of GFP for flow visualization and chromatographic process development.

Low-cost microbioreactor for high-throughput bioprocessing.

The design of a microbioreactor is described. An optical sensing system was used for continuous measurements of pH, dissolved oxygen, and optical density in a 2 mL working volume. The K(L)a of the microbioreactor was evaluated under different conditions. An Escherichia coli fermentation in both the microbioreactor and a standard 1 L bioreactor showed similar pH, dissolved oxygen, and optical density profiles.%The low cost of the microbioreactor, detection system, and the small volume of the fermentation broth provide a basis for development of a multiple-bioreactor system for high-throughput bioprocess optimization.

Polarization-based sensing of glucose using an oriented reference film.

We describe a new approach to glucose sensing using polarization measurements in the presence of a stretch-oriented reference film. The method relies on measurement of the polarized emission from the reference film and of a fluorophore which changes intensity in response to glucose. A glucose-sensitive fluorescent signal was provided by the glucose/galactose binding protein from E. coli. This protein was labeled with an environmentally sensitive fluorophore at a single genetically inserted cysteine residue, and displayed decreased fluorescence upon glucose binding. Using the protein and the reference film we observed glucose-sensitive polarization values for micromolar glucose concentrations. This method of polarization-based sensing is generic and can be used for any sensing fluorophore which displays a change in intensity. In principle, one can construct simple and economical devices for this type of glucose measurement. © 1999 Society of Photo-Optical Instrumentation Engineers.

Steam-sterilizable, fluorescence lifetime-based sensing film for dissolved carbon dioxide.

An autoclavable sensing film was developed for monitoring dissolved CO2. The sensing film, based on fluorescence resonance energy transfer (FRET), consisted of a fluorescent donor, an acceptor, and a quaternary ammonium hydroxide, which were doped in a two-component silicone film. As no aqueous solution was used in the sensing film matrix, the sensing film was unaffected by osmotic pressure. Fluorescence lifetime was selected as the sensing parameter, and measured in frequency domain using phase fluorometry. Upon exposure to 20% CO2-saturated water, a 43 degrees increase in phase angle was observed at 100 MHz. The process was fully reversible when the sensing film was exposed to nitrogen-saturated water. The estimated response and recovery times for 90% signal change were 1 min (for a step change from 0 to 6.7% CO2-saturated water) and 1.5 min (for a step change from 6.7 to 3.3% CO2-saturated water). When used for on-line monitoring of dissolved CO2 produced by a culture of Escherichia coli, the sensing film showed a similar trend to that obtained from off-line measurements using a wet chemistry analyzer.

Green fluorescent protein as a real time quantitative reporter of heterologous protein production.

Since its cloning and commercial availability, applications of green fluorescent protein (GFP) as a reporter gene have become prevalent in many aspects of science. The attributes of GFP could also be applied to the area of heterologous protein production. The work described here represents the first experiments to use GFP as a generic tool to monitor protein production in bioprocess development. We have constructed a plasmid containing an operon fusion of the two reporter genes GFP and chloramphenicol acetyl transferase (CAT). CAT served as a "model" recombinant protein product to demonstrate the in situ quantifiable reporting mechanism of GFP. Our results indicate there is a direct correlation between the fluorescence intensity of GFP and the functional activity of the downstream CAT protein. In addition, there is a quantitative relationship between the level of CAT protein concentration and GFP fluorescence. These experiments provide the groundwork for using GFP as an in situ reporter gene for scale-up and process optimization of recombinant protein production.

On-line green fluorescent protein sensor with LED excitation.

We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time.

Noninvasive oxygen measurements and mass transfer considerations in tissue culture flasks.

Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were monitored. Oxygen levels were measured noninvasively by interrogating an oxygen-sensitive patch mounted on the interior surface of the tissue culture flask with an optrode from outside the tissue culture flask. Readings were made in tissue culture flasks with caps both cracked open and completely closed. Although the oxygen in the gas phase remained near atmospheric oxygen levels in both flasks, over time the liquid-phase oxygen tension at the bottom of the flasks reached zero during cell growth in both the open and closed tissue culture flasks. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and probably unnecessary.The mass transfer characteristics of the tissue culture flask were also studied. The dominant resistance to oxygen mass transfer to the sensor and the cells was through the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone. This suggests that mixing at a microscale occurs. Volumetric and specific oxygen consumption rates were also calculated from the sensor data. These consumption rates were comparable with values published elsewhere. (c) 1996 John Wiley & Sons, Inc.

Formation of DNA-protein cross-links in cultured mammalian cells upon treatment with iron ions.

Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon treatment with iron or copper ions was investigated. Cultured murine hybridoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin samples were isolated from treated and control cells. Analyses of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization revealed a significant increase over the background amount of 3-[(1,3-dihydrio-2,4-dioxopyrimidin-5-yl)-methyl]- L-tyrosine (Thy-Tyr crosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with ascorbic acid or coincubation with dimethyl sulfoxide did not significantly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to detect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associated with free radical reactions.

DNA base modifications and membrane damage in cultured mammalian cells treated with iron ions.

We investigated DNA base damage in mammalian cells exposed to exogenous iron ions in culture. Murine hybridoma cells were treated with Fe(II) ions at concentrations of 10 microM, 100 microM, and 1 mM. Chromatin was isolated from treated and control cells and analyzed by gas chromatography/mass spectrometry for DNA base damage. Ten modified DNA bases were identified in both Fe(II)-treated and control cells. The quantification of modified bases was achieved by isotope-dilution mass spectrometry. In Fe(II)-treated cells, the amounts of modified bases were increased significantly above the background levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M in the culture medium did not significantly inhibit the formation of modified DNA bases. A mathematical simulation used to evaluate the plausibility of DNA damage upon Fe(II) treatment predicted a dose-dependent response, which agreed with the experimental results. In addition, Fe(II) treatment of cells increased the cell membrane permeability and caused production of lipid peroxides. The nature of DNA base lesions suggests the involvement of the hydroxyl radical in their formation. The failure of dimethyl sulfoxide to inhibit their formation indicates a site-specific mechanism for DNA damage with involvement of DNA-bound metal ions. Fe(II) treatment of cells may increase the intracellular iron ion concentration and/or cause oxidative stress releasing metal ions from their storage sites with subsequent binding to DNA. Identified DNA base lesions may be promutagenic and play a role in pathologic processes associated with iron ions.