Faculty of Science - Mahidol University
Faculty of Science - Mahidol University
Faculty of Science - Mahidol University
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286<br />
Many eukaryotic proteins have been produced successfully in<br />
Escherichia coli. However, not every gene can be expressed<br />
efficiently in this organism. Most proteins, especially those with<br />
multiple disulfide bonds, have been shown to form insoluble protein<br />
or inclusion body in E. coli. An inactive form <strong>of</strong> protein would<br />
require an in vitro refolding step to regain biological functions. In<br />
this study, we described the system for soluble expression <strong>of</strong> a singlechain<br />
variable fragment (scFv) against hepatocellular carcinoma<br />
(Hep27scFv) by coexpressing Dsb protein and enhancing with<br />
medium additives. The results revealed that overexpression <strong>of</strong><br />
DsbABCD protein showed marked effect on the soluble production<br />
<strong>of</strong> Hep27scFv, presumably facilitating correct folding. The optimal<br />
condition for soluble scFv expression could be obtained by adding<br />
0.5M sorbitol to the culture medium. The competitive enzyme-linked<br />
immunosorbent assay (ELISA) indicated that soluble scFv expressed<br />
by our method retains binding activity toward the same epitope on<br />
a hepatocellular carcinoma cell line (HCC-S102) recognized by intact<br />
antibody (Ab) (Hep27 Mab). Here, we report an effective method<br />
for soluble expression <strong>of</strong> scFv in E. coli by the Dsb coexpression<br />
system with the addition <strong>of</strong> sorbitol medium additive. This method<br />
might be applicable for high-yield soluble expression <strong>of</strong> proteins<br />
with multiple disulfide bonds.<br />
(Published in Biotechnol Bioeng 2005; 91: 418-24. Supported by<br />
<strong>Science</strong> and Technology Incubation Program in Advanced Region,<br />
Japan <strong>Science</strong> and Technology Corporation.)<br />
CONSTRUCTION OF SCFV DERIVED FROM A<br />
TUMOR-ASSOCIATED MONOCLONAL ANTIBODY<br />
HAVING TUMORICIDAL ACTIVITY ON HUMAN<br />
HEPATOCELLULAR CARCINOMA. (NO. 740)<br />
Tungpradabkul S 1 , Sandee D 1 , Puthong S 2 , Laohathai K 2 .<br />
1 Department <strong>of</strong> Biochemistry, <strong>Faculty</strong> <strong>of</strong> <strong>Science</strong>, <strong>Mahidol</strong><br />
<strong>University</strong>, Bangkok; 2 Institute <strong>of</strong> Biotechnology and Genetic<br />
Engineering, Chulalongkorn <strong>University</strong>, Bangkok.<br />
Key words : single-chain variable fragment, tumor-associated<br />
monoclonal antibody, tumoricidal effect.<br />
A mouse monoclonal antibody (Mab-HepTAA43),<br />
classified as an anti-tumor-associated antigen, was raised by<br />
immunizing BALB/c mice with the Thai human hepatocellular<br />
carcinoma S102 (HCC-S102) cell line cells using hybridoma<br />
techniques. The Mab-HepTAA43 reacted with and markedly<br />
inhibited the growth <strong>of</strong> human hepatocellular carcinoma cell lines<br />
as well as a tumor mass in an animal model. Human hepatoma<br />
transplanted into nude mice did not show metastasis after 20<br />
injections amounting to a total <strong>of</strong> about 4mg <strong>of</strong> the Mab over 1month<br />
period. A single-chain variable fragment (scFv molecule<br />
derived from the Mab was constructed by phage display method.<br />
DNA sequence analysis <strong>of</strong> the active variable regions <strong>of</strong> both heavyand<br />
light-chains <strong>of</strong> the cDNA clone was subsequently performed.<br />
The scFv43 molecule contains a V(L) kappa type and a unique V(H)<br />
sequence having 88% amino acid homology to that <strong>of</strong> Mab-MAK B<br />
raised against tumor-associated antigen. Immunohistochemical<br />
staining on frozen sections <strong>of</strong> paired hepatoma (NCI-I) and normal<br />
liver tissue from the same individual showed that both scFv43 and<br />
Mab-HepTAA43 antibodies reacted with hepatoma but not with<br />
normal liver tissue. The results suggest that scFv43 may be useful<br />
in the immunotherapy <strong>of</strong> hepatocellular carcinoma.<br />
(Published in Mol Immunol 2005; 42: 713-9. Supported by <strong>Mahidol</strong><br />
<strong>University</strong>, Chulalongkorn <strong>University</strong>; Thailand National <strong>Science</strong><br />
and Technology Development Agency.)<br />
PURIFICATION AND CHARACTERISATION OF<br />
KERATINASE FROM A THERMOTOLERANT<br />
FEATHER-DEGRADING BACTERIUM. (NO. 741)<br />
Suntornsuk W 1 , Tongjun J 1 , Onnim P 1 , Oyama H 2 ,<br />
Ratanakanokchai K 3 , Kusamran T 4 , Oda K 2 .<br />
1 Department <strong>of</strong> Microbiology, <strong>Faculty</strong> <strong>of</strong> <strong>Science</strong>, King<br />
Mongkut’s Institute <strong>of</strong> Technology Thonburi, Bangkok;<br />
2 Department <strong>of</strong> Applied Biology, <strong>Faculty</strong> <strong>of</strong> Textile <strong>Science</strong>,<br />
Kyoto Institute <strong>of</strong> Technology, Kyoto, Japan; 3 Department <strong>of</strong><br />
Biochemical Technology, School <strong>of</strong> Bioresources and Technology,<br />
King Mongkut’s Institute <strong>of</strong> Technology Thonburi, Bangkok;<br />
4 Department <strong>of</strong> Biochemistry, <strong>Faculty</strong> <strong>of</strong> <strong>Science</strong>, <strong>Mahidol</strong><br />
<strong>University</strong>, Bangkok.<br />
<strong>Faculty</strong> <strong>of</strong> <strong>Science</strong><br />
Key words : Bacillus licheniformis, keratinase, thermotolerance<br />
Isolation and identification <strong>of</strong> a thermotolerant featherdegrading<br />
bacterial strain from Thai soil as well as purification and<br />
properties <strong>of</strong> its keratinase were investigated. The thermotolerant<br />
bacterium was identified as Bacillus licheniformis. The keratinase<br />
was purified to homogeneity by three-step chromatography. The<br />
purified enzyme exhibited a high specific activity (218 U mg -1 ) with<br />
86-fold purification and 25% yield. The enzyme was monomeric<br />
and had a molecular mass <strong>of</strong> 35 kDa. The optimum pH and<br />
temperature for the enzyme were 8.5 and 60°C, respectively. The<br />
enzyme activity was significantly inhibited by PMSF and partly<br />
inhibited by EDTA and iodoacetamide, but was stimulated by metal<br />
ions. It hydrolysed soluble proteins with a relative activity <strong>of</strong> 4–<br />
100% and insoluble proteins, including keratins, with a relative<br />
activity <strong>of</strong> 3–35%. Therefore, the enzyme could improve the<br />
nutritional value <strong>of</strong> meat- and poultry-processing wastes containing<br />
keratins, collagen and gelatin.<br />
(Published in World J Microbiol Biotech 2005; 21: 1111–<br />
7.Supported by Japan Society for Promotion <strong>of</strong> <strong>Science</strong> and National<br />
Research Council <strong>of</strong> Thailand.)<br />
QUINONE REDUCTASE INDUCERS IN<br />
AZADIRACHTA INDICA A. JUSS FLOWERS,<br />
AND THEIR MECHANISMS OF ACTION. (NO. 742)<br />
Sritanaudomchai H 1 , Kusamran T 1 , Kuakulkiat W 2 ,<br />
Bunyapraphatsara N 2 , Hiransalee A 3 , Tepsuwan A 4 , Kusamran<br />
WR 4 .<br />
1 Department <strong>of</strong> Biochemistry, <strong>Faculty</strong> <strong>of</strong> <strong>Science</strong>, <strong>Mahidol</strong><br />
<strong>University</strong>, Bangkok; 2 Department <strong>of</strong> Pharmacognosy, <strong>Faculty</strong><br />
<strong>of</strong> Pharmacy, <strong>Mahidol</strong> <strong>University</strong>, Bangkok; 3 Department <strong>of</strong><br />
Medical <strong>Science</strong>, Ministry <strong>of</strong> Public Health, Bangkok; 4 Chemical<br />
Carcinogenesis Section, Research Division, National Cancer<br />
Institute, Bangkok.<br />
We have previously shown that the flowers <strong>of</strong> neem tree<br />
(Azadirachta indica A. Juss, family Meliaceae), Thai variety,<br />
strongly induced the activity <strong>of</strong> glutathione S-transferase (GST) while<br />
resulting in a significant reduction in the activities <strong>of</strong> some<br />
cytochrome P(450)-dependent monooxygenases in rat liver, and<br />
possess cancer chemopreventive potential against chemicallyinduced<br />
mammary gland and liver carcinogenesis in rats. In the<br />
present study, 2 chemicals possessing strong QR inducing activity<br />
were fractionated from neem flowers using a bioassay based on the<br />
induction <strong>of</strong> QR activity in mouse hepatoma Hepa 1c1c7 cultured<br />
cells. Spectroscopic characteristics revealed that these compounds<br />
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