Institut Penyelidikan Perubatan Molekul - Tesis

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Comparative Analysis Of Nucleic Acid Residues In Core Streptavidin Using Three Isolation Techniques
(2024-08)
Alias, Nurul Nadia Mohamad
Recombinant core streptavidin (cSAV) is a truncated non-glycosylated tetrameric SAV which has been widely utilised in a wide range of biotechnological applications. Due to cSAV commercial importance, recombinant cSAV has been extensively produced using expression host system such as Escherichia coli to high expression level. Nevertheless, accumulation of cSAV in high levels results in the formation of insoluble non-functional inclusion bodies (IBs) which requires efficient downstream processing steps to overcome. This process involves isolation of IBs from cell lysates through combination of cell disruption techniques, multiple centrifugation steps, IBs solubilization and refolding to acquire correctly refolded cSAV. However, during IBs preparation, presence of cellular contaminants such as residual nucleic acids is inevitable and can affect subsequent refolding process. Hence, in this study, the IBs isolation process from our previous group (Process A) was improved to obtain high quality cSAV IBs in an effort to attain refolded cSAV with minimal contaminants. The improvements were enhanced with the incorporation of quantitative polymerase chain reactions (qPCR) for residual DNAs monitoring. By employing combined cell disruption approaches such as extensive sonication (Process B) and addition of benzonase nuclease (Process C), cSAV IBs with 99% removal of residual DNAs were achieved. A 10% increment of cSAV refolding yield (72%) and 83% reduction of residual DNAs from refolding of 1 mg cSAV IBs were observed under extensive sonication. Despite perceiving the least residual DNAs, refolding of cSAV was found not affected and the activity of refolded cSAV was not compromised.
Identification Of Immunogenic Proteins From Cysts And Trophozoites Of Giardia Lamblia By Immunoassay
(2024-07)
Roshidi, Norhamizah
Giardia lamblia exists in two stages in the life cycle; the motile trophozoite and the resistant cyst that is highly adapted to harsh environments, infecting humans and animals with a disease known as giardiasis. Giardiasis is a significant public health concern, with an estimated 280 million symptomatic human infections occurring each year and contributing to 1.6 million cases of diarrheal death in 2016. In Malaysia, cases of giardiasis reportedly ranged from 2.6% to 25% from 1970 to 2000 and rise to 10.4% to 28.3% from 2002 to 2019. However, studies on giardiasis are very limited and mostly focused on the variation of gene expression and vaccine development. Therefore, this study was conducted to characterize Giardia proteins that may lead to a better understanding of Giardia-immunogenic proteins potential for diagnostics and therapeutic applications. The approach of this study involves in-vitro cultivation of G. lamblia trophozoites and cyst in different culture medium, protein extraction and separation using OFFGEL™ as an improved pre-fractionation step prior to immunoassays and mass-spectrometry analysis to identify the identity of potential immunogenic proteins. The maximum number of trophozoites of 9.6 x 106 cell/ml was successfully obtained in the TYI-S-33 medium at pH 7 for 72 hours at 37°C, while the maximum number of cysts of 7.45 x 105 cell/ml was produced in complete modified TYI-S-33 medium containing 0.25 mg/ml of porcine bile at pH 7.8 for 72 hours at 37°C.
Development Of Leishmania Tarentolae Expression Systems For Different Recombinant Antibody Formats
(2024-04)
Jing Yi, Lai
Production of recombinant antibodies has been an important topic for biomedical applications. Currently, expression of monoclonal antibodies is mainly performed using mammalian cell lines such as Chinese hamster ovary (CHO) cells and Human embryonic kidney 293 (HEK293) cells due the advantage of human-like post-translational modifications. However, the use of mammalian cell lines has some drawbacks especially in term of cost. The introduction of Leishmania tarentolae as an expression host is viewed as an interesting alternative due to their post-translational modification, which is similar to human but is more cost-effective for expression and maintenance. In this study, the use of L. tarentolae was explored as a platform to express recombinant antibodies in scFv-Fc and IgG format. A single chain fragment variable (scFv) clone against long-chain neurotoxin (LNTX) of Naja kaouthia was identified from naïve human antibody library. The clone was expressed and characterized in Escherichia coli as scFv and further converted to scFv-Fc and IgG format for expression in L. tarentolae. The expression of IgG was achieved using a bicistronic vector system. A total of two constructs, encompassing both possible position of the heavy chain (HC) and light chain (LC), were expressed. The construct with the HC-LC gene arrangement was found to perform better in term of yield. The stability of the IgG at 26ºC and 45ºC was also examined. While the expression performance of antibody clone is dependent on the antibody gene sequence, the study shows the potential application of L. tarentolae for expressing both scFv-Fc and IgG. In conclusion, the L. tarentolae expression system can be touted as a possible alternative system for the expression of scFv-Fc and IgG antibody clones.
Phage Display Of Naïve Human T-Cell Receptors In Single Chain Format Against Isocitrate Lyase (Icl) And Tumor Necrosis Factor Alpha (Tnfα)
(2014-04)
Ch’Ng Chiew Wen, Angela
T-cell receptors (TCRs) bind with peptide-MHC or lipid-CD1 complexes while antibodies directly bind to antigen. The binding region of T-cell receptors (TCRs) and antibodies exhibit similar three-dimensional (3D) structures. Both composed of two chains and each chain contains two folded domains of one variable and one constant which associate together to create the antigen binding site between them. Since both antibodies and TCR have similar structures, there exists a potential for TCRs to serve as a binding scaffold to bind full protein antigens, much like antibodies. Consequently, the entire experimental study was designed with the aim of investigating whether TCRs can indeed bind to full antigen proteins just like how antibodies bind to antigen proteins. The recognition site of the TCR comprises of the alpha and beta variable regions. Consequently, both alpha and beta variable regions are interconnected through a glycine-serine linker, resulting in the formation of a single-chain TCR. Additionally, a naïve scTCR library was meticulously crafted using phage display technology with a library size of 1010. Then, the scTCR library was used to isolate scTCR binders against two designated antigens recombinant isocitrate lyase (rICL) and recombinant tumor necrosis factor alpha (rTNFα). A total of three monoclonal scTCR binders to rICL and two to rTNFα respectively were isolated. All of the human antibody constant (CH1 or CK) scTCR fusion proteins expressed best at 30 °C except a-rICL-2G10 scTCR.
Development And Characterization Of Monoclonal Antibodies Against Ancylostoma Caninum Ancylostoma-Secreted Protein 5 (Asp5) By Phage Display
(2024-07)
Brenda Song, Pei Chui
Parasitic nematodes such as Ancylostoma caninum engages in a complex life cycle, in which it can impact the hosts at its various stages of development and infection. During infective stage, Ancylostoma caninum releases a multitude of proteins to modulate the host’s immune response, which ensuring its survival within the host environment. Among these proteins, Ancylostoma-secreted protein 5 (ASP5) is critically involved in the parasite-host interaction such as the immunomodulation, tissue invasion and facilitating the blood feeding process, hence making it a promising target for intervention strategies aimed at controlling hookworm infections among canines. By harnessing phage display technology, this study aims to develop monoclonal antibodies targeting Ancylostoma-secreted protein 5 (ASP5), by employing the human naïve scFv antibody library through in vitro biopanning. The library was subjected to a total of three rounds of panning against 10 μg of ASP5 antigen, with progressively stringent washing conditions (10, 20 and 30 washes respectively) to isolate the clones. This study delves into Ancylostoma-secreted protein 5 as an antigenic candidate for monoclonal antibody development in which it was expressed using the bacterial strain of BL21(DE3) and purified with Immobilized Metal Affinity Chromatography (IMAC) purification method using the nickel-charged affinity resin. The developed antibody was characterized on its antibody-antigen interactions, including cross-reactivity and binding strength via Enzyme-linked immunosorbent assay (ELISA).

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