Hepatitis B poses a major public
health problem worldwide with approximately 350 million carriers
of the virus in the world. According to the Malaysian Liver Foundation,
close to six per cent of Malaysians could be carriers of this
silent killer. Patients have an increased risk of developing chronic
liver diseases such as cirrhosis and hepatocellular carcinoma.
The foundation has encouraged Malaysians to take precautionary
measures like regular blood tests and vaccination against the
virus in an effort to curtail its development on the Malaysian
public. Alarmed by the dramatic rise of Hepatitis B cases in Malaysia,
Dr Tan Wen Siang, an Associate Professor at Universiti Putra Malaysia
(UPM), has focused his research on developing therapeutic agents
such as inhibitors and monoclonal antibodies against Hepatitis
B Virus using phage display technology.
The phage display
technology is a tool used to screen and select for proteins on
the basis of binding or molecular recognition. It refers to the
display of functional foreign proteins on the surface of bacteriophage
(also commonly referred to as a phage). They are parasitic bacterium
which infects only bacteria. Dr Tan’s research projects
concentrate on the use of M13 phage, also known as filamentous
phage due to its long physical appearance, which infects Escherichia
coli.
When asked why the M13 is preferred, Dr Tan reasoned that
“because it is a single-stranded DNA (ssDNA) phage,
which can be easily cloned.” DNA normally exists in
two complementary strands that form a double helix, but
it can be separated into two strands of ssDNA as shown in
Figure 1. Separation becomes necessary
when genes are being transcribed or when DNA is replicated
prior to cell division.
Successful insertion of a foreign sequence to the gpIII
gene results in the corresponding peptides to be displayed
as fusion proteins at the tip of the filamentous phage,
identified as the gpIII protein in Figure 2.
Figure 1:
Replication of DNA
Figure
2: A schematic representative of M13 phage
The
displayed foreign proteins on the tip of the phage can then
be used as unique markers to attract and bind specific proteins
of interest (Figure 3). When such proteins
are ‘captured’, they can then be washed, amplified
and sequenced. They can then be identified using a range
of standard laboratory techniques to allow for better understanding
of its role in the specified disease.
Figure
3: Binding of specific proteins
of interest to the displayed peptides
“Since molecular recognition
plays a vital role in many biological processes, ranging from
drug-target, hormone-receptor, enzyme-substrate and antibody-antigen
interactions, this technique thus has the potential to be used
in drug discovery, antibody engineering, vaccine development and
so on,” Dr Tan commented. “Drugs can be discovered
by identifying the small molecules that inhibit the propagation
of viruses and microorganisms using phage display technology”.
When comparing the production
of monoclonal antibodies using the conventional method and the
described phage display technology, Dr Tan reasoned that “the
conventional method to produce monoclonal antibodies using animal
cells is more expensive and time-consuming. In contrast to the
conventional method, phage display technology does not involve
the fusion of spleen cells and myeloma cells, which is technically
tedious and difficult. Moreover, the phage display system can
select antibodies more efficiently than through the conventional
system, not to mention that it is cheaper to produce antibodies
using bacteria, rather than mammalian cell lines”.
Hepatitis B virus
(HBV), for instance, poses a major public health problem worldwide.
For chronically infected patients, synthetic alpha-interferon
(a protein produced by the immune system in response to a viral
infection) and synthetic nucleoside analogs (artificial copies
of a nucleoside; when incorporated into a virus DNA or RNA during
viral replication, the nucleoside analogs act to prevent production
of new viruses) appears to be the best hope in combating the virus.
However, according to Dr Tan, the successful rate of these treatments
is less than 50%.
Therefore, development of an effective
therapeutic agent will ultimately provide an additional mean to
control and eradicate the disease. Inhibitors that may be useful
as therapeutic agents to control HBV have been successfully identified
in one of Dr Tan’s research projects, done in collaboration
with Prof. Sir Kenneth Murray from University of Edinburgh and
Dr Mike Dyson from University of Cambridge. The phage display
library has been employed to select the molecules that inhibit
the assembly of HBV. In a related project, Dr Tan, together with
Prof Datin Khatijah Yusoff and Prof Seow Heng Fong, both from
UPM, are working to generate and design monoclonal antibodies
against HBV using filamentous phages. These recombinant phages
have the potential to be further developed into diagnostic reagents.
Dr Tan has high hopes and sees
great potential in his research projects, particularly the ones
on HBV and Nipah virus. He believes that phage display technology
is a key technique that has become a very useful tool in his quest
to help prevent, control and cure a range of diseases that collectively
accounts for millions of lives worldwide.
In the words
of the late Professor Albert Szent-Gyorgyi, a Nobel Prize winner,
“Discovery consists of seeing what everybody has seen and
thinking what nobody has thought”. This example of innovative
thinking from researchers like Dr Tan has shown us that novel
solutions, borne by the fusion of different ideas, can provide
practical answers in solving mankind’s problems.
Dr.
Tan Wen Siang is an Associate Professor at the Department
of Microbiology, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia. He obtained his BSc
(Hons) in Biochemistry and Microbiology, and MSc in Molecular
Biology from UPM. He did his PhD at the University of Edinburgh
under the supervision of Prof. Sir Kenneth Murray, who developed
the first effective recombinant vaccine for hepatitis B.
Together with his colleagues and students, he has published
42 papers in refereed journals and 135 papers in scientific
proceedings, symposia, conferences and seminars. He is also
the inventor for 6 patents pending. He has been awarded
many prizes, including The Ministry of Science, Technology
and Environmental Award (1993), ICI Gold Medal (1993), Chemorpharm
Award (1993), Gold Medal in I-Tex (2003), and Gold Medal
in Expo Science and Technology (2003). His biography was
recorded in Who’s Who in Medicine and Healthcare,
5th Editions 2004-2005. He is a chairman and also a member
of the supervisory committee for 9 PhD and 22 MSc students.
