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Tour
of the Le Grice Laboratory
Biofermentation
Projects currently underway and recently completed have required
HIV-1 RT in amounts varying from a few nanograms, for evaluation
of enzymatic activity, to several hundred milligrams for X-ray
crystallography and titration microcalorimetry. Moreover,
enzymes such as T7
RNA polymerase, Taq DNA polymerase, and T4 RNA ligase are
required in substantial quantities for routine manipulations
in several projects. Large-scale biofermentation facilities
thus serve a central role in each theme currently under investigation.
In order to meet these demands, the RT Biochemistry Section
has invested in a dedicated biofermentation facility. The
figure on the left illustrates a New Brunswick Bioflo 4500
5-20 liter, self-sterilizing, computer-controlled fermentor/
bioreactor operated by Dr. Miller.
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With controlled aeration, pH, and dissolved oxygen, these reaction
vessels permit growth of our E. coli strains to optical
densities exceeding 30, which provides us with ~300 gm of biomass
from a 10-liter fermentation in ~12 hours. Working at this scale
also requires an efficient procedure for rapid harvesting of
the biomass. To achieve this,
a Hereaus Contifuge T7 Stratos continuous-action centrifuge
is connected directly to the biofermentor. This approach allows
us to harvest the 10-liter reaction volume in less than 45 minutes.
In addition to large-scale RT production for biophysical analysis,
several retroviral and retrotransposon enzymes are currently
under investigation, where purification in the 1-5 mg range
is sufficient. For these needs, the laboratory is equipped with
three independently controlled Innova platform shakers (right),
each of which is capable of holding up to 6 2-liter culture
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Large-Scale
Protein Purification
While biofermentation provides us with the necessary quantities
of biomass, this must be complemented with efficient and rapid
protein purification methodologies. A dedicated cold laboratory
contains HPLC/FPLC, FPLC, and low-pressure chromatographic equipment
to meet these needs (left). For each instrument, a selection
of affinity, ion exchange, and gel permeation matrices are available
in both analytical and preparative scales. However, as the quantity
of biomass increases, the use of chromatographic techniques
at early purification steps becomes impractical and necessitates
the application of batch strategies, which are likewise conducted
in the cold laboratory. A second workbench in the cold laboratory
is reserved for low-temperature electrophoresis when fractionating
nucleic acid duplexes by nondenaturing polyacrylamide gel electrophoresis. |
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Oligonucleotide and Peptide Separation
Several projects recently initiated in the laboratory involve
purification of nucleic acids and peptides by HPLC. Examples
of this include the synthesis of oligonucleotides containing
proteolytic,
nucleolytic, and photoactivable bioconjugates. In addition,
modified bases such asinosine and 2-aminopurine are being incorporated
into nucleic acids to probe the structure of retroviral reverse
transcription complexes. Finally, in conjunction with mass spectrometry,
proteolytic fragments of p66/p51 HIV-1 RT are separated by HPLC
for analysis by microsequencing. For such projects, the laboratory
makes use of a Beckman/Coulter System Gold HPLC equipped with
a diode array, fluorescence detector, and column oven (right).
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Molecular Modeling
Our structure/function studies with HIV-1 RT have been aided by
the availability of several high-resolution crystal structures of
the p66/p51 enzyme. These include (a) the unliganded
enzyme,
(b) co-crystals with several nonnucleoside-based inhibitors, (c) a binary
complex containing duplex DNA, (d) a ternary complex of enzyme,
DNA, and an incoming deoxynucleoside triphosphate, and (e) an RNA-DNA
hybrid encompassing the polypurine tract. Molecular modeling thus
plays a pivotal role in interfacing our biochemical studies on mutant
enzymes with the consequences for the structure of either the p66
or p51 subunit. In addition, using a combination of biochemistry
and molecular modeling, we are investigating several small-molecule
inhibitors of RNase H function that have been identified recently.
Researchers in the RT Biochemistry Section receive instruction on
molecular modeling using a Silicon Graphics Octane 2 workstation
with staff of the Advanced Biomedical Computing Center (left).
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Protein Footprinting via Mass Spectrometry
A variety of chemical and enzymatic probes are available to study changes in nucleic acid conformation upon binding of ligand. However, solution methodologies providing high-resolution information on the protein component of this complex are considerably less developed and have traditionally relied on tagging the protein at its N- or C-terminus with an epitope or
phosphorylation site. Mass spectroscopy is rapidly emerging as a simple yet highly precise approach that can be applied to this problem. Nucleoprotein complexes are subjected to hydrolysis by one of several highly specific endoproteinases, after which the products are
resolved by MALDI-TOF. The high degree of accuracy of the mass spectrometer thereafter allows the assignment of peaks to peptides of the original protein. In collaboration with the NCI Laboratory of Medicinal Chemistry, researchers of the RT Biochemistry Section make use of a Kratos Kopmact SEQ mass spectrometer equipped with a 1.7-meter flight tube for enhanced resolution (right). Peptide mixtures analyzed by the mass spectrometer can also be verified by their separation via HPLC and peptide sequencing.
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Laser
Crosslinking
Bioconjugation (i.e., site-specific tethering of two molecules
to generate a novel complex displaying the combined properties
of its individual components) is a powerful complement
to
high-resolution crystallographic and spectroscopic
methods in providing structural information on protein-nucleic
acid complexes. Projects currently underway in the laboratory
involve attachment of artificial nucleases and proteases
to the protein and nucleic acid components of nucleoprotein
complexes. In addition, site-specific attachment of photocrosslinking
agents provides high-resolution structural information
on the interaction of RT with its conformationally distinct
substrates. In order to probe the interaction of RT with
template and primer nucleotides in real time (i.e., in
the millisecond range). The laboratory makes use of a
New Wave "Tempest" pulse laser in combination
with a Kin-Tek rapid-quench apparatus (left).
Laser crosslinking is also in use to evaluate the binding
site of small-molecule RNase H antagonists.
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In Vivo Studies with HIV and FIV
Although projects in the laboratory have to this stage been primarily of a biochemical nature, studies involving culture of HIV and feline immunodeficiency virus (FIV) have recently been initiated. These include control of reverse transcription during initiation of (-) strand DNA
synthesis in FIV and evaluation of central termination in HIV. In vitro experimentation suggests that a novel interaction of tRNALys,3 and the FIV genome controls initiation, and efforts are underway to study this long-range intermolecular interaction through alterations of the FIV genome. A second project involves the specificity of cPPT/CTS elements, which are proposed to play a structural role at a late step in the HIV reverse transcription cycle. Similarity between these elements of HIV and equine infectious anemia virus is under investigation. For such studies, the laboratory makes use of cell culture facilities (right) in the laboratory of Dr. Vineet KewalRamani.
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Centrifugal
Filtration
During large-scale protein purification, exchanging buffers
and desalting macromolecular solutions via dialysis become
both impractical and time consuming. Centrifugal filtration,
using
filtration devices with a variety of membrane cut-offs,
permits the rapid concentration of solutions in volumes
ranging from 100 ml (using filters inserted into Eppendorf
microcentrifuge tubes) to 1000 ml, where 200-ml devices
are used in combination with high-speed tabletop centrifuges
(left). Low adsorbtion of the cellulose
membrane and the components of the filtration device combine
to give recoveries in excess of 95% with a processing
time of 10-60 minutes. |
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Specialized Oligonucleotide Synthesis
Introducing modified nucleosides
into synthetic DNA and RNA oligonucleotides by phosphoramidite
chemistry allows us to examine in considerably more detail
the interaction of RT with the structurally distinct nucleic
acid duplexes encountered during replication. These substrates
include duplex DNA, duplex RNA, and RNA/DNA hybrids. Our
recent studies have involved introducing non-hydrogen-bonding
pyrimidine isosteres (or shape mimics) into DNA to evaluate
how the flexibility of DNA/RNA hybrids influences their
recognition by the RTs of HIV-1 (Rausch et al., PNAS,
2003) and the Saccharomyces cerevisiae LTR-retrotransposon
Ty3 (Lener et al., JBC, 2003). Another study has taken
advantage of the unique fluorescence properties of the
cytidine analog, pyrrolo-dC, whose emission spectrum is
considerably removed from that of tryptophan, to explore
hydrogen-bonding patterns in RNA/DNA hybrids (Dash et
al., NAR, 2004, in press). Lastly, the unique properties of certain
nucleoside analogs can be exploited to study the conformation
of RNA/DNA hybrids by NMR spectroscopy. This combination
of biophysical and biochemical studies clearly requires
multi-milligram quantities of both DNA and RNA oligonucleotides
containing modified bases. Synthesis of specialized oligonucleotides
is conducted by individual researchers of the RT Biochemistry
Section, each of whom has received training from Dr. Yi-Brunozzi
(right), a specialist in RNA synthesis. |
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Nonradioactive
Electrophoretic Methods: Capillary Electrophoresis
Conventionally,
analyzing the enzymatic activities of RT has required
5' or 3' end-labeling of DNA or RNA oligonucleotides
with [32P], resolving reaction products by
high-resolution denaturing gel electrophoresis, and
their quantitation by densitometry or phosphorimaging.
Substituting radioactive procedures with colorimetric
or fluorescent assays, while at the same time allowing
the same sample throughput, would clearly be advantageous.
Using phosphoramidite chemistry, fluorescent tags such
as fluorescein can be introduced within or at the termini
of both DNA and RNA. Following RNase H-mediated hydrolysis
or polymerase-mediated DNA synthesis, the reaction products
can be separated by capillary electrophoresis and quantified
by laser-induced fluorescence. The instrument illustrated
(left) will resolve RNase H-derived hydrolysis
products in 5-7 minutes, and at the some time provides
direct quantitation and single-nucleotide separation.
In addition, its 96-well platform allows multiple samples
to be loaded and analyzed overnight. In collaboration
with researchers of the NCI Molecular Targets Development
Program and SAIC Analytical Chemistry Laboratory, capillary
electrophoresis plays a central role in high-throughput
screening of inhibitors of HIV-1 and human RNase H.
Additional projects of the RT Biochemistry Section involve
adapting the technology for both steady-state and pre-steady-state
analysis of the RTs of HIV-1 and Ty3.
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When direct visualization of reaction products is not necessary (i.e., when simply the extent of RNase H-mediated hydrolysis must be measured), a variation of the fluorescence approach
can again be used. In this case, the RNA and DNA components of an RNA/DNA
hybrid contain a fluorescence donor and quencher, respectively,
the proximity of which results in fluorescence quenching.
Following RNase H-mediated hydrolysis, the fluorescence
donor is relieved of the quenching environment, resulting
in a simple, sensitive, and quantifiable "off/on" RNase
H assay. Using a fluorescence plate reader, samples can
once more be evaluated in a 96-well format (right).
The two RNase H assays described briefly here, in conjunction
to DNA polymerase assays under development, represent
considerable savings in both time and cost, as well as
reducing the environmental burden associated with storage
and disposal of radioactive waste. |
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Last
modified: 22 December 2008
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