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Performance
of 50 Completed ATP Projects
Status
Report - Number 2
NIST SP 950-2
Chapter
3 - Biotechnology
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Amersham
Pharmacia Biotech
(formerly U.S. Biochemical Corporation)
Searching for New Enzymes in
Deep-Sea Microorganisms
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| Found
in every living organism, enzymes are naturally produced proteins
that regulate the rate of chemical reactions occurring in cells. Enzymes
are responsible for many thousands of biochemical processes and are
vital for cell growth and the production and use of energy within
cells. Enzymes also play a vital role in industrial applications and
microbiology research. By providing the foundation for many of the
advances in biotechnology, enzymes are now revolutionizing health
care and agriculture industries. |
COMPOSITE
PERFORMANCE SCORE
(Based on a four star rating.)
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Unlocking Genetic
Information
The current biotechnology revolution began only a few decades ago when
scientists discovered they could use certain enzymes to unlock the genetic
information contained in a cell’s chromosomes. Found in the genes
of DNA strands, a sequence of nucleotides provide cells with instructions
on how to build the proteins and amino acids that are used to carry out
biological functions. Study of this sequence information has already led
to major advances in cloning, forensic identification, and cancer research.
It may also lead to therapeutic treatment and custom drug design for other
diseases currently without a cure.
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| Deep Sea Vent
"Black Smoker." |
Thermally Stable Enzymes
Needed for DNA Amplification
The techniques used to obtain sequencing information involve a series
of processes to tear apart a cell and break open its components to reveal
the underlying genes. This is done with the help of an enzyme called DNA
polymerase. DNA polymerase also plays a critical role in another important
genetic process that is used to create multiple copies of a known sample
of DNA. This process, called amplification, works by placing an isolated
sample of DNA in a solution of bases, primers, and DNA polymerase. The
solution is then heated, which causes the double-stranded DNA to separate
into two single strands. The mixture is then cooled so the primers can
bind to pre-established sites along each of the separate DNA strands.
When the mixture is reheated, the DNA polymerase moves along the single
strands of DNA and attaches the bases to form two identical, double strands
of DNA. Repeating the heating and cooling cycle doubles the strands, and
after about 30 cycles, the single fragment of DNA is sufficiently amplified
to provide enough material for sequencing and further research.
The enzymes initially
used for sequencing and amplification were not thermally stable and tended
to break down when subjected to the heat necessary to separate the DNA
strands. As a result, the polymerization process required constant monitoring
to insure that adequate enzyme amounts were present.
Scientists discovered
that microbes inhabiting the hot springs of Yellowstone Park produce a
heat tolerant DNA polymerase. Although this enzyme, commercially developed
as “Taq,” was thermally stable for amplification of DNA, it
often proved inaccurate for DNA sequencing. As the problems with Taq became
apparent, the search for superior enzymes became a major quest.
ATP Supports the Search
for a More Effective Enzyme
In 1993, U.S. Biochemical Corporation (USB) applied to ATP to study the
commercial potential of enzymes extracted from newly discovered microorganisms
found living in the superheated waters of thermal vents on the deep ocean
floor. Researchers hoped that microorganisms living in such extreme temperatures
could be used to produce an enzyme that was both thermally stable and
more accurate than Taq.
At the time, USB,
a small company located in Cleveland, Ohio, was a recognized leader in
the development of DNA sequencing products and had more than 20 years
of experience in the biochemical industry. Shortly after originating this
project, USB was purchased by U.K.-based Amersham International. Amersham
pledged full commitment to this project using USB’s original plan
and U.S.-based personnel and facilities. The ATP awarded $1.6 million
to USB, and determined that continuation of the project after the company
was purchased by Amersham International would be in the U.S. economic
interest.
Working together with
the University of Maryland’s Center of Marine Biotechnology (COMB),
Amersham’s initial objective was to collect a large number of exotic
hyperthermophiles from the superheated waters found in thermal vents along
the ocean floor. Hyperthermophiles actually thrive in extremely high temperatures
(80°–110°C), high pressures (@3300l bps), and highly caustic
solutions made up of sulfur and other minerals.
The unusual properties
that make hyperthermophiles so appealing also make them extremely difficult
and expensive to work with. The researchers designed special equipment
and improved methods to allow the tasks of isolating and purifying the
new enzymes to be carried out in an environment that matches their deep-sea
habitat. Once the new enzymes were available, researchers screened them
to determine how their properties compare to Taq.
As the work developed,
researchers found that the DNA polymerases from deep-sea hyperthermophiles,
like Pyrococcus furiosus, did not outperform Taq. Indeed, none of the
enzymes they produced was found to be superior to Taq. As researchers
continued their study, they began to realize that the search for the Holy
Grail of enzymes was not yielding results from the ocean depths.
Scientific Discovery
Leads Amersham To Redirect Research Efforts
Two years after the project began, a discovery published in the Proceedings
of the National Academy of Sciences by Tabor and Richardson showed how
scientists could re-engineer Taq to achieve greater fidelity and accuracy
of sequence data.(1)
By creating a single amino acid change in a polymerase, Tabor and Richardson
showed that they could affect the enzyme’s ability to discriminate
among nucleotides, and thereby produce uniform sequence signals. This
important discovery meant that the structure of an enzyme such as Taq
could be reengineered to provide the discrimination functions that lead
to accurate, uniform sequence signal intensity. It wasn’t the Holy
Grail, but it was very close.
So profound was this
discovery that Amersham immediately stopped its effort to screen deep-sea
hyperthermophilic enzymes and set about to reproduce the work of Tabor
and Richardson. They soon discovered that these results could not be replicated
with hyperthermophilic enzymes, but they could reengineer other thermophilic
enzymes to produce properties superior to Taq.
Faster Development
of ThermoSequenase
With the knowledge that naturally occurring hyperthermophilic enzymes
were not viable alternatives to reengineered thermophilic enzymes, Amersham
licensed Tabor and Richardson’s technique and produced Thermo-Sequenase,
a DNA polymerase that is both thermostable and produces amplified DNA
sequences of uniform signal intensity. (2)
With ATP’s support, the development of ThermoSequenase was advanced
by at least six months. Former director of the Human Genome Project, Dr.
David Smith of the Department of Energy, singles out the timely development
of ThermoSequenase in 1995 as being critical to the Human Genome Project,
stating, “We would be in deep trouble if [such technologies] were
at a less mature stage of development. (3)
ThermoSequenase is
now incorporated into Amersham’s leading line of sequencing reagent
kits. Currently, these kits account for sales of over $15 million per
year and are expected to reach sales of $60 million in 2000.
Researchers using
ThermoSequenase for DNA sequencing now obtain 10 to 25 percent more information
from each sequencing experiment. The availability of ThermoSequenase has
effectively reduced the cost of sequencing substantially. It has also
enabled greater use of advanced automated sequencing machines that can
now operate without the need for constant monitoring of enzyme amounts.
Customers of services using ThermoSequenase benefit from more accurate
and more efficient sequencing. Development of ThermoSequenase also has
stimulated competition in the enzyme market and has improved the quality
of enzymes in biotechnology applications.
A New Field of Research
Bears Fruit in Unexpected Ways
ATP’s cofunded project with Amersham has been praised as one of the
first federally supported efforts to explore the potential of newly discovered
deep-sea life. This has opened up a new field of research that was completely
unknown two decades ago. (4)
The Amersham researchers
developed methods and applied them to search deep-sea life for an enzyme
that offered a thermally stable and more accurate means of DNA sequencing.
It did not find the hoped-for sequencing enzyme in the deep sea; in fact,
during the course of the project, Amersham was able to conclude that hyperthermophiles
were not the answer to the search for a better polymerase enzyme for DNA
sequencing. The company quickly took a different approach to solving the
problem. The project helped to position Amersham and its academic collaborators
so that they could take advantage of new emerging techniques in enzyme
reengineering. Pioneering use of these techniques led to accelerated development
of ThermoSequenase. Hence, the project achieved its goal, but not in the
expected way. And, it did find a useful enzyme in the deep sea, though
not the one of central focus.
Amersham has effectively
diffused knowledge gained through the project by issuing 16 journal publications
and a number of patents. The company filed for seven U.S. patents, five
of which had been granted at the time of this study. In turn, the development
of ThermoSequenase, and the release of information about it, have led
to greater market competition, and encouraged the development of competing
enzymes.
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Project
Highlights
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PROJECT:
To study deep-sea microorganisms in an effort to identify, isolate,
and characterize their commercially important enzymes for use in
life sciences research, including a suitable enzyme for DNA sequencing,
and industrial applications.
Duration: 2/15/1994 – 2/14/1997
ATP Number: 93-01-0113
FUNDING (in
thousands):
| ATP |
$1,558
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65%
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| Company |
839
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35%
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| Total |
$2,397
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ACCOMPLISHMENTS:
Recent advances in molecular biology and discoveries of exotic new
life forms have created commercial opportunities in the fields of
genomics and enzyme development. The ATP-funded projeect led by
Amersham explored the properties of newly discovered, heat-loving
microorganisms with the objective of advancing the scientific knowledge
of their genetic makeup and identifying the commercial potential
of their expressed enzymes.
Key accomplishments
for this project include:
- isolation
and genomic characterization of hyperthermophiles obtained from
deep-sea thermal vent fluids;
- successful
partial DNA sequencing of hyperthermophiles using
directed DNA cloning and other advanced sequencing techniques;
- successful
cloning of genes encoding enzymes that have unique applications
in life science research and are of significant commercial interest.
These
enzymes include DNA polymerases used in DNA cycle sequencing and
several modifying enzymes used to improve the
detection, selection and manipulation of DNA sequences;
- publication
of sequencing results as “A Survey of the Genome of the Hyperthermophilic
Archaeon Pyrococcus Furiosus” in the first volume of Genome
Science and Technology, one of 16 publications which helped to
diffuse knowledge gained through the project;
- applied for
7 patents and, by the time of this study, had been granted 5 patents:
- "Thermostable
alkaline phosphatase of thermus thermophilus" (No. 5,633,138:
filed 5/30/1995, granted 5/27/97);
- "Thermostable
DNA polymerase from thermoanaerobacter thermohydrosulfuricus"
(No. 5,744,312: filed 12/13/1996, granted 4/28/1998);
- "Thermostable
DNA polymerases" (No. 5,885,813: filed 5/14/1996, granted 5/23/1999);
- "Modified
Pol-II type DNA polymerases" (No. 5,827,716: filed 7/30/1996,
granted 10/27/1998);
- "Proteins
from pyrococcus furiosus" (No. 5,719,056: filed 4/26/1996, granted
2/17/1998).
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CITATIONS
BY OTHERS OF PROJECT’S PATENTS:
See Figure 3.1.
COMMERCIALIZATION STATUS:
From its research on hyperthermophiles, Amersham developed a thermally
stable alkaline phosphatase with applications in the detection of
genetic diseases. This product has reached sales of $1.2 million
per year. With the application of newly discovered enzyme reengineering
techniques, Amersham accelerated development of ThermoSequenase,
a DNA polymerase that is both thermostable and accurate for DNA
sequencing. Sales of ThermoSequenase are $15 million per year and
are expected to reach $60 million by 2000. Enzymes to replace chemical
catalysts in large-scale industrial applications have yet to be
developed.
OUTLOOK:
Amersham completed many of the technical objectives of this project
and accelerated research and development of new enzymes for use
in DNA diagnostics. Although it successfully isolated at least one
commercially useful enzyme, it did not achieve the ultimate goal
of finding a superior hyperthermophilic enzyme. Nevertheless, the
project is at least partially responsible for accelerated development
of a thermally stable DNA polymerase, ThermoSequenase. This enzyme
has helped to revolutionize automated DNA cycle sequencing by providing
improved accuracy in fluorescent readouts of sequence information.
Benefits are accruing from a growing number of health care and diagnostic
applications that rely on accurate and timely DNA sequence information.
The potential application areas are numerous, including medical
diagnostics, gene therapy, drug discovery, human therapeutics, cell
cloning, cancer genetics, agricultural biotechnology, forensic identification,
toxicology, and environmental monitoring.
The development
of competing enzymes is underway and there is substantial market
competition in this area. The outlook is promising that the considerable
knowledge gained from this project may yet lead to the development
of new classes of industrial enzymes.
Composite
Performance Score:
COMPANY:
Amersham Pharmacia Biotech
(Formerly U.S. Biochemical Corporation, then Amersham International)
800 Centennial Avenue
Piscataway, NJ 08855
Contact: Carl W. Fuller
Phone: (732) 457-8000
Informal Collaborator: University of Maryland’s Center
of Marine Biotechnology
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____________________
1. S.
Tabor and C. Richardson, Proceedings of the National Academy of Sciences,
92, pp. 6339-6343, 1995.
2. At the start of the project, Amersham was producing
and marketing a DNA polymerase known as Sequenase that could produce accurate,
uniform sequencing. However, this product was not thermally stable and
could not compete with Taq when used in cycle sequencing machines.
3. The Seventh International Genome Sequencing and
Analysis Conference, September 1995, available on the Internet (www.ol-nl.cov/TecllResources/Hulllall
Genome/publicat/97pr/evolve.html).
4. William J. Brode, The Universe Below: Discovering
the Secrets of the Deep Sea, Touchstone: Simon & Schuster, 1997, p.
283.
Return to Table
of Contents or go to next section.
Date created: April
2002
Last updated:
April 12, 2005
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