Displaying publications 1 - 20 of 61 in total

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  1. A 35 kDa antigenic protein from Shigella flexneri: in silico structural and functional studies
    Yung-Hung RL, Ismail A, Lim TS, Choong YS
    Biochem Biophys Res Commun, 2011 Nov 18;415(2):229-34.
    PMID: 21982766 DOI: 10.1016/j.bbrc.2011.09.116
    Shigella flexneri serotype 2a is a major public health concern in the developing and under-developed countries which contributes to shigellosis endemic and mortality. Thus, there is an urgent need for a rapid diagnostic test for effective therapy and disease management. Previous study showed that a ∼35 kDa antigenic protein from S. flexneri is a potential biomarker. We therefore modelled the three-dimensional structure of the antigen to probe its functionality which could aid in the development of an antigen-based diagnostic. Results showed that the antigen is a transmembrane protein consists of OmpA and OmpA-like domains. The OmpA domain is a beta-barrel embedded in the outer membrane with four surface-exposed extracellular loops. The OmpA-like domain is linked to the OmpA domain with a 17 amino acids linker and located in the periplasmic. Docking of peptidoglycan into the groove of OmpA-like domain might help in catalyzing the bacterial cell wall formation. Both domains are expected to be involved in the virulence, structural stability, pathogenesis and survival of Shigella thus made the 35 kDa protein a suitable shigellosis diagnostic biomarker. This structural elucidation will also enable a better identification of the epitope regions for the development of specific binders to the 35 kDa antigen.
  2. Elucidating isoniazid resistance using molecular modeling
    Wahab HA, Choong YS, Ibrahim P, Sadikun A, Scior T
    J Chem Inf Model, 2009 Jan;49(1):97-107.
    PMID: 19067649 DOI: 10.1021/ci8001342
    The continuing rise in tuberculosis incidence and the problem of drug resistance strains have prompted the research on new drug candidates and the mechanism of drug resistance. Molecular docking and molecular dynamics simulation (MD) were performed to study the binding of isoniazid onto the active site of Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA) in an attempt to address the mycobacterial resistance against isoniazid. Results show that isonicotinic acyl-NADH (INADH) has an extremely high binding affinity toward the wild type InhA by forming stronger interactions compared to the parent drug (isoniazid) (INH). Due to the increase of hydrophobicity and reduction in the side chain's volume of A94 of mutant type InhA, both INADH and the mutated protein become more mobile. Due to this reason, the molecular interactions of INADH with mutant type are weaker than that observed with the wild type. However, the reduced interaction caused by the fluctuation of INADH and the mutant protein only inflected minor resistance in the mutant strain as inferred from free energy calculation. MD results also showed there exists a water-mediated hydrogen bond between INADH and InhA. However, the bridged water molecule is only present in the INADH-wild type complex, reflecting the putative role of the water molecule in the binding of INADH to the wild type protein. The results support the assumption that the conversion of prodrug isoniazid into its active form INADH is mediated by KatG as a necessary step prior to target binding on InhA. Our findings also contribute to a better understanding of INH resistance in mutant type.
  3. Fulltext Vaccines for TB: Lessons from the Past Translating into Future Potentials
    Tye GJ, Lew MH, Choong YS, Lim TS, Sarmiento ME, Acosta A, et al.
    J Immunol Res, 2015;2015:916780.
    PMID: 26146643 DOI: 10.1155/2015/916780
    Development of vaccines for infectious diseases has come a long way with recent advancements in adjuvant developments and discovery of new antigens that are capable of eliciting strong immunological responses for sterile eradication of disease. Tuberculosis (TB) that kills nearly 2 million of the population every year is also one of the highlights of the recent developments. The availability or not of diagnostic methods for infection has implications for the control of the disease by the health systems but is not related to the immune surveillance, a phenomenon derived from the interaction between the bacteria and their host. Here, we will review the immunology of TB and current vaccine candidates for TB. Current strategies of developing new vaccines against TB will also be reviewed in order to further discuss new insights into immunotherapeutic approaches involving adjuvant and antigens combinations that might be of potential for the control of TB.
  4. Fulltext Theoretical investigation on structural, functional and epitope of a 12 kDa excretory-secretory protein from Toxoplasma gondii
    Tommy YB, Lim TS, Noordin R, Saadatnia G, Choong YS
    BMC Struct Biol, 2012 Nov 27;12:30.
    PMID: 23181504 DOI: 10.1186/1472-6807-12-30
    BACKGROUND: Toxoplasma gondii is an intracellular coccidian parasite that causes toxoplasmosis. It was estimated that more than one third of the world population is infected by T. gondii, and the disease is critical in fetuses and immunosuppressed patients. Thus, early detection is crucial for disease diagnosis and therapy. However, the current available toxoplasmosis diagnostic tests vary in their accuracy and the better ones are costly.

    RESULTS: An earlier published work discovered a highly antigenic 12 kDa excretory-secretory (ES) protein of T. gondii which may potentially be used for the development of an antigen detection test for toxoplasmosis. However, the three-dimensional structure of the protein is unknown. Since epitope identification is important prior to designing of a specific antibody for an antigen-detection based diagnostic test, the structural elucidation of this protein is essential. In this study, we constructed a three dimensional model of the 12 kDa ES protein. The built structure possesses a thioredoxin backbone which consists of four α-helices flanking five β-strands at the center. Three potential epitopes (6-8 residues) which can be combined into one "single" epitope have been identified from the built structure as the most potential antibody binding site.

    CONCLUSION: Together with specific antibody design, this work could contribute towards future development of an antigen detection test for toxoplasmosis.

  5. Optimisation of human VH domain antibodies specific to Mycobacterium tuberculosis heat shock protein (HSP16.3)
    Soong JX, Chan SK, Lim TS, Choong YS
    J Comput Aided Mol Des, 2019 03;33(3):375-385.
    PMID: 30689080 DOI: 10.1007/s10822-019-00186-z
    Mycobacterium tuberculosis (Mtb) 16.3 kDa heat shock protein 16.3 (HSP16.3) is a latency-associated antigen that can be targeted for latent tuberculosis (TB) diagnostic and therapeutic development. We have previously developed human VH domain antibodies (dAbs; clone E3 and F1) specific against HSP16.3. In this work, we applied computational methods to optimise and design the antibodies in order to improve the binding affinity with HSP16.3. The VH domain antibodies were first docked to the dimer form of HSP16.3 and further sampled using molecular dynamics simulation. The calculated binding free energy of the HSP16.3-dAb complexes showed non-polar interactions were responsible for the antigen-antibody association. Per-residue free energy decomposition and computational alanine scanning have identified one hotspot residue for E3 (Y391) and 4 hotspot residues for F1 (M394, Y396, R397 and M398). These hotspot residues were then mutated and evaluated by binding free energy calculations. Phage ELISA assay was carried out on the potential mutants (E3Y391W, F1M394E, F1R397N and F1M398Y). The experimental assay showed improved binding affinities of E3Y391W and F1M394E against HSP16.3 compared with the wild type E3 and F1. This case study has thus showed in silico methods are able to assist in optimisation or improvement of antibody-antigen binding.
  6. Fulltext Broad specificity of immune helminth scFv library to identify monoclonal antibodies targeting Strongyloides
    Rahumatullah A, Balachandra D, Noordin R, Baharudeen Z, Lim YY, Choong YS, et al.
    Sci Rep, 2021 01 28;11(1):2502.
    PMID: 33510342 DOI: 10.1038/s41598-021-82125-3
    Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.
  7. Fulltext Development of an antigen-DNAzyme based probe for a direct antibody-antigen assay using the intrinsic DNAzyme activity of a daunomycin aptamer
    Omar N, Loh Q, Tye GJ, Choong YS, Noordin R, Glökler J, et al.
    Sensors (Basel), 2013;14(1):346-55.
    PMID: 24379042 DOI: 10.3390/s140100346
    G-Quadruplex (G-4) structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays.
  8. Generation and selection of naïve Fab library for parasitic antigen: Anti-BmSXP antibodies for lymphatic filariasis
    Omar N, Hamidon NH, Yunus MH, Noordin R, Choong YS, Lim TS
    Biotechnol Appl Biochem, 2018 May;65(3):346-354.
    PMID: 28833498 DOI: 10.1002/bab.1591
    Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 109 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis.
  9. Antibody Phage Display
    Nur A, Schubert M, Lai JY, Hust M, Choong YS, Isa WYHW, et al.
    Methods Mol Biol, 2023;2702:3-12.
    PMID: 37679612 DOI: 10.1007/978-1-0716-3381-6_1
    The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.
  10. Application of Computational Techniques in Antibody Fc-Fused Molecule Design for Therapeutics
    Ng CL, Lim TS, Choong YS
    Mol Biotechnol, 2024 Apr;66(4):568-581.
    PMID: 37742298 DOI: 10.1007/s12033-023-00885-x
    Since the advent of hybridoma technology in the year 1975, it took a decade to witness the first approved monoclonal antibody Orthoclone OKT39 (muromonab-CD3) in the year 1986. Since then, continuous strides have been made to engineer antibodies for specific desired effects. The engineering efforts were not confined to only the variable domains of the antibody but also included the fragment crystallizable (Fc) region that influences the immune response and serum half-life. Engineering of the Fc fragment would have a profound effect on the therapeutic dose, antibody-dependent cell-mediated cytotoxicity as well as antibody-dependent cellular phagocytosis. The integration of computational techniques into antibody engineering designs has allowed for the generation of testable hypotheses and guided the rational antibody design framework prior to further experimental evaluations. In this article, we discuss the recent works in the Fc-fused molecule design that involves computational techniques. We also summarize the usefulness of in silico techniques to aid Fc-fused molecule design and analysis for the therapeutics application.
  11. Fulltext Synthesis, Biological Evaluation and Molecular Docking of Novel Indole-Aminoquinazoline Hybrids for Anticancer Properties
    Mphahlele MJ, Mmonwa MM, Aro A, McGaw LJ, Choong YS
    Int J Mol Sci, 2018 Jul 31;19(8).
    PMID: 30065164 DOI: 10.3390/ijms19082232
    A series of indole-aminoquinazolines was prepared via amination of the 2-aryl-4-chloroquinazolines with the 7-amino-2-aryl-5-bromoindoles. It was then evaluated for cytotoxicity in vitro against human lung cancer (A549), epithelial colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma (C3A), breast adenocarcinoma (MCF-7), and cervical cancer (HeLa) cells. A combination on the quinazoline and indole moieties of a 2-phenyl and 2-(4-fluorophenyl) rings in compound 4b; 2-(4-fluorophenyl) and 3-chlorophenyl rings in compound 4f; or the two 2-(4-fluorophenyl) rings in compound 4g, resulted in significant and moderate activity against the Caco-2 and C3A cell lines. The indole-aminoquinazoline hybrids compounds 4f and 4g induced apoptosis in Caco-2 and C3A cells, and were also found to exhibit moderate (IC50 = 52.5 nM) and significant (IC50 = 40.7 nM) inhibitory activity towards epidermal growth factor receptor (EGFR) against gefitinib (IC50 = 38.9 nM). Molecular docking suggests that 4a⁻h could bind to the ATP region of EGFR like erlotinib.
  12. Fulltext Benzofuran-appended 4-aminoquinazoline hybrids as epidermal growth factor receptor tyrosine kinase inhibitors: synthesis, biological evaluation and molecular docking studies
    Mphahlele MJ, Maluleka MM, Aro A, McGaw LJ, Choong YS
    J Enzyme Inhib Med Chem, 2018 Dec;33(1):1516-1528.
    PMID: 30274538 DOI: 10.1080/14756366.2018.1510919
    A series of 2-arylbenzo[b]furan-appended 4-aminoquinazoline hybrids were prepared and evaluated for cytotoxicity in vitro against the human lung cancer (A549), colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma (C3A) and cervical cancer (HeLa) cell lines. Compounds 10d and 10j exhibited significant cytotoxicity against the C3A and Caco-2 cell lines and induced apoptosis in these cell lines. Likewise, compounds 10d and 10e exhibited significant inhibitory activity towards epidermal growth factor receptor-tyrosine kinase phosphorylation (IC50 values of 29.3 nM and 31.1 nM, respectively) against Gefitinib (IC50 = 33.1 nM). Molecular docking of compounds 10 into EGFR-TK active site suggests that they bind to the region of EGFR like Gefitinib does. [Formula: see text].
  13. Biological evaluation the 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones as potential dual α-glucosidase and α-amylase inhibitors with antioxidant properties
    Mphahlele MJ, Magwaza NM, Malindisa ST, Choong YS
    Chem Biol Drug Des, 2021 08;98(2):234-247.
    PMID: 34013660 DOI: 10.1111/cbdd.13893
    The 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones (azaborininone) were synthesized as analogues of the 2-arylquinazoline-4-ones and screened through enzymatic assay in vitro for inhibitory effect against α-glucosidase and α-amylase activities. These azaborininones exhibited moderate to good inhibitory effect against these enzymes compared to acarbose used as a reference standard. The results are supported by the enzyme-ligand interactions through kinetics (in vitro) and molecular docking (in silico) studies. The test compounds also exhibited significant antioxidant activity through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. These azaborininone derivatives exhibited no effect on the viability of the human lung cancer (A549) cell line after 24 hr and were also not toxic towards the Vero cells.
  14. Fulltext Synthesis and In Vitro Cytotoxicity of the 4-(Halogenoanilino)-6-bromoquinazolines and Their 6-(4-Fluorophenyl) Substituted Derivatives as Potential Inhibitors of Epidermal Growth Factor Receptor Tyrosine Kinase
    Mphahlele MJ, Paumo HK, Choong YS
    Pharmaceuticals (Basel), 2017 Nov 20;10(4).
    PMID: 29156606 DOI: 10.3390/ph10040087
    Series of the 2-unsubstituted and 2-(4-chlorophenyl)-substituted 4-anilino-6-bromoquinazolines and their 6-(4-fluorophenyl)-substituted derivatives were evaluated for in vitro cytotoxicity against MCF-7 and HeLa cells. The 2-unsubstituted 4-anilino-6-bromoquinazolines lacked activity, whereas most of their 2-(4-chlorophenyl) substituted derivatives were found to exhibit significant cytotoxicity and selectivity against HeLa cells. Replacement of bromine with 4-fluorophenyl group for the 2-unsubstituted 4-anilinoquinazolines resulted in superior activity against HeLa cells compared to Gefitinib. The presence of a 4-fluorophenyl group in the 2-(4-chlorophenyl) substituted derivatives led to increased cytotoxicity against HeLa cells, except for the 3-chloroanilino derivative. The most active compounds, namely, 3g, 3l, and 4l, were found to exhibit a moderate to significant inhibitory effect against epidermal growth factor receptor tyrosine kinase (EGFR-TK). The EGFR molecular docking model suggested that these compounds are nicely bound to the region of EGFR.
  15. Fulltext Synthesis and Evaluation of N-(3-Trifluoroacetyl-indol-7-yl) Acetamides for Potential In Vitro Antiplasmodial Properties
    Mphahlele MJ, Mmonwa MM, Choong YS
    Molecules, 2017 Jul 02;22(7).
    PMID: 28671598 DOI: 10.3390/molecules22071099
    A series of novel N-((2,5-diaryl-3-trifluoroacetyl)-1H-indol-7-yl)acetamides has been prepared via a successive and one-pot reaction sequence involving initial trifluoroacetic acid-mediated Beckmann rearrangement of the oximes derived from the 1-(2,5-diaryl-1H-indol-7-yl)ethanones, followed by trifluoroacetylation of the incipient N-(2,5-diaryl-1H-indol-7-yl)-acetamides with trifluoroacetic anhydride. The prepared compounds were evaluated for potential in vitro antiplasmodial properties. Preliminary results from antiplasmodial activity against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum revealed that a combination of 2-(4-flurophenyl)- and 5-(4-fluorophenyl) or 2-(4-flurophenyl)- and 4-fluorostyryl groups in compounds 3(a,f) and 4(a,g), for example, is required for biological activity for both series of compounds. Their possible mode of action against the plasmodial parasite is explained theoretically through molecular docking of the most active compounds against the parasite lactate dehydrogenase (pLDH). These compounds were docked at the entrance of NAD+ in pLDH presumably hindering entry of lactate to cause the observed inhibition effect of pLDH. The four compounds were found to exhibit low toxicity against monkey kidney Vero cells at the highest concentrations tested.
  16. Fulltext Synthesis, In Vitro Evaluation and Molecular Docking of the 5-Acetyl-2-aryl-6-hydroxybenzo[b]furans against Multiple Targets Linked to Type 2 Diabetes
    Mphahlele MJ, Choong YS, Maluleka MM, Gildenhuys S
    Biomolecules, 2020 03 07;10(3).
    PMID: 32156083 DOI: 10.3390/biom10030418
    The 5-acetyl-2-aryl-6-hydroxybenzo[b]furans 2a-h have been evaluated through in vitro enzymatic assay against targets which are linked to type 2 diabetes (T2D), namely, α-glucosidase, protein tyrosine phosphatase 1B (PTP1B) and β-secretase. These compounds have also been evaluated for antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging method. The most active compounds against α-glucosidase and/or PTP1B, namely, 4-fluorophenyl 2c, 4-methoxyphenyl 2g and 3,5-dimethoxyphenyl substituted 2h derivatives were also evaluated for potential anti-inflammatory properties against cyclooxygenase-2 activity. The Lineweaver-Burk and Dixon plots were used to determine the type of inhibition on compounds 2c and 2h against α-glucosidase and PTP1B receptors. The interactions were investigated in modelled complexes against α-glucosidase and PTP1B via molecular docking.
  17. Fulltext Synthesis, Structure, Carbohydrate Enzyme Inhibition, Antioxidant Activity, In Silico Drug-Receptor Interactions and Drug-Like Profiling of the 5-Styryl-2-Aminochalcone Hybrids
    Mphahlele MJ, Agbo EN, Choong YS
    Molecules, 2021 May 04;26(9).
    PMID: 34064448 DOI: 10.3390/molecules26092692
    The 2-amino-5-(3/4-fluorostyryl)acetophenones were prepared and reacted with benzaldehyde derivatives to afford the corresponding 5-styryl-2-aminochalcone hybrids. The trans geometry of the styryl and α,β-unsaturated carbonyl arms, and the presence of NH…O intramolecular hydrogen bond were validated using 1H-NMR and X-ray data. The 2-amino-5-styrylacetophenones and their 5-styryl-2-aminochalcone derivatives were screened in vitro for their capability to inhibit α-glucosidase and/or α-amylase activities. Their antioxidant properties were evaluated in vitro through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. Kinetic studies of the most active derivatives from each series against α-glucosidase and/or α-amylase activities have been performed supported by molecular docking studies to determine plausible protein-ligand interactions on a molecular level. The key aspects of the pharmacokinetics of these compounds, i.e., absorption, distribution, metabolism, and excretion have also been simulated at theoretical level. The most active compounds from each series, namely, 2a and 3e, were evaluated for cytotoxicity against the normal monkey kidney cells (Vero cells) and the adenocarcinomic human epithelial (A549) cell line to establish their safety profile at least in vitro.
  18. Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology
    Mohd Ali MR, Sum JS, Aminuddin Baki NN, Choong YS, Nor Amdan NA, Amran F, et al.
    Int J Biol Macromol, 2021 Jan 31;168:289-300.
    PMID: 33310091 DOI: 10.1016/j.ijbiomac.2020.12.062
    Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually varied, allowing it to be mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of a specific agent in clinical samples is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody against rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.
  19. Assembly of highly diverse genes using degenerate oligonucleotides by temperature cascade
    Loh Q, Bahara NH, Choong YS, Lim TS
    Anal Biochem, 2012 Dec 1;431(1):54-6.
    PMID: 22975202 DOI: 10.1016/j.ab.2012.08.025
    The quality of a nucleotide-based library such as a synthetic antibody library is highly dependent on the diversity available. Diversity can be generated using degenerate oligonucleotides introduced during gene assembly. Conventional approaches to gene assembly are not efficient for oligonucleotides with long stretches of degeneracy. We propose an efficient alternative for simultaneous introduction of three randomized regions in a synthetic antibody gene via temperature cascading. The strategy takes advantage of DNA reannealing kinetics. The strategy can be adopted for generating diversity of gene inserts during the construction of nucleotide-based libraries.
  20. Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system
    Loh Q, Leong SW, Tye GJ, Choong YS, Lim TS
    Anal Biochem, 2015 May 15;477:56-61.
    PMID: 25769419 DOI: 10.1016/j.ab.2015.02.026
    The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.
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