Displaying publications 1 - 20 of 33 in total

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  1. Fulltext Integrase deficient lentiviral vector: prospects for safe clinical applications
    Yew CT, Gurumoorthy N, Nordin F, Tye GJ, Wan Kamarul Zaman WS, Tan JJ, et al.
    PeerJ, 2022;10:e13704.
    PMID: 35979475 DOI: 10.7717/peerj.13704
    HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector's replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.
    Matched MeSH terms: Genetic Vectors/genetics
  2. Dual-vector nanoengineered mRNA-CRISPR delivery systems for precision gene therapy in choroidal melanoma
    Thakur SK, Malviya R, Chandra P, Sridhar SB, Shareef J, Wadhwa T
    Nanomedicine (Lond), 2026 Nov;21(21):3013-3032.
    PMID: 42965688 DOI: 10.1080/17435889.2026.2754253
    Choroidal melanoma is the most common primary intraocular malignancy, and effective gene therapy remains limited by ocular barriers, tumor heterogeneity, and inefficient nucleic acid delivery. This review critically analyses the molecular rationale, engineering principles, emerging nanoarchitectures, biological validation, and translational prospects of dual-vector nanoengineered mRNA-CRISPR delivery systems for precision gene therapy in choroidal melanoma. The manuscript also highlights key challenges and future strategies for clinical implementation. Current evidence indicates that dual-vector platforms enable independent optimization of mRNA and CRISPR cargos while maintaining coordinated spatiotemporal therapeutic activity. We conducted a literature search in PubMed, Scopus, and Web of Science for relevant studies published from January 2015 to September 2026. Functional cargo partitioning, vector modularity, programmable intracellular trafficking, and ocular-targeted nanoengineering improve delivery precision and reduce biological compromise associated with co-encapsulation. Lipid, polymeric, hybrid, biomimetic, and viral-nonviral architectures each provide distinct engineering advantages depending on therapeutic objectives. Preclinical studies support enhanced genome editing, transient protein restoration, improved tumor suppression, and reduced off-target effects. Nevertheless, manufacturing scalability, regulatory standardization, long-term safety, and clinical validation remain major barriers to translation. Dual-vector nanoengineered mRNA-CRISPR systems represent a promising next-generation platform for precision gene therapy in choroidal melanoma by enabling coordinated and programmable therapeutic delivery. Continued advances in modular nanoengineering, personalized cargo selection, and clinically scalable manufacturing will be essential for successful translation into precision ocular oncology.
    Matched MeSH terms: Genetic Vectors/genetics
  3. Fulltext Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX
    Hassan MI, McSorley FR, Hotta K, Boddy CN
    J Vis Exp, 2017 06 27.
    PMID: 28715370 DOI: 10.3791/55187
    Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.
    Matched MeSH terms: Genetic Vectors/genetics*
  4. Large BACs transfect more efficiently in circular topology
    Wong YC, Osahor A, Al-Ajli FOM, Narayanan K
    Anal Biochem, 2021 10 01;630:114324.
    PMID: 34363787 DOI: 10.1016/j.ab.2021.114324
    The effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells.
    Matched MeSH terms: Genetic Vectors/genetics
  5. Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells
    Wong YC, Ng AWR, Chen Q, Liew PS, Lee CW, Sim EUH, et al.
    ACS Synth Biol, 2023 Apr 21;12(4):909-921.
    PMID: 37026178 DOI: 10.1021/acssynbio.2c00580
    Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.
    Matched MeSH terms: Genetic Vectors/genetics
  6. Vector-Mediated Cancer Gene Therapy Reduces Toxicity and Inhibition of Lung Carcinoma Growth in Nude Mice
    Khalaf AT, Wan J, Wei H, Fubing S, Zainol J, Kadir SYA, et al.
    Appl Biochem Biotechnol, 2024 Jan;196(1):261-274.
    PMID: 37119504 DOI: 10.1007/s12010-023-04463-4
    Replication-competent oncolytic adenovirus (TOA2) gene therapy is a recently introduced anti-tumor treatment regimen with superior results. The biodistribution studies of virus vector-based medicine seem more cautious and have been given much attention recently in terms of its quality and safety in preclinical trials. The current study determined the biodistribution and safety of a replication-competent adenovirus in different organs to predict its toxicity threshold. The present study has used TOA2, while biodistribution analysis was performed in human lung carcinoma A549-induced tumor-bearing nude mice model. Intratumoral injection was applied onto tumor-bearing mice with the adenovirus (3×1010 VP per mouse). Mice were sacrificed at the end of the experiment and the organs were dissected. Biodistribution analysis was done with complete hexon gene detection in each organ using quantitative real-time polymerase chain reaction (qRT-PCR). The biodistribution and concentration profiles showed that the TOA2 is well distributed in the entire tumor tissue. After dose 3 at day 11, the concentration of the virus has increased in the tumor tissue from 2240.54 (± 01.69) copies/100 ng genome to 13,120.28 (± 88.21) copies/100 ng genome on the 18th day, which eventually approached 336.45 (± 23.41) copies/100ng genome on the day 36. On the contrary, the concentration of the same decreased in the order of the liver, kidney, spleen, lung, and heart over time but no distributional traces in gonads. But the concentration found decreased dramatically in blood and other organs, while at the end of the experiment no detectable distribution was seen besides tumor tissue. The study confirms that adenovirus-based tumor therapy using conditionally replicating competent oncolytic TOA2 exhibited great efficiency with no toxicity at all.
    Matched MeSH terms: Genetic Vectors/genetics
  7. Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis
    Osahor A, Deekonda K, Lee CW, Sim EU, Radu A, Narayanan K
    Anal Biochem, 2017 10 01;534:46-48.
    PMID: 28693990 DOI: 10.1016/j.ab.2017.07.008
    Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.
    Matched MeSH terms: Genetic Vectors/genetics
  8. Fulltext Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
    Koko I, Song AA, Masarudin MJ, Abdul Rahim R
    BMC Biotechnol, 2019 11 27;19(1):82.
    PMID: 31775775 DOI: 10.1186/s12896-019-0575-x
    BACKGROUND: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system.

    RESULTS: The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA.

    CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

    Matched MeSH terms: Genetic Vectors/genetics
  9. Improved DNA Delivery Efficiency of Bacterial Vectors by Co-Delivery with Exogenous Lipid and Antimicrobial Reagents
    Osahor AN, Narayanan K
    Methods Mol Biol, 2021;2211:15-27.
    PMID: 33336267 DOI: 10.1007/978-1-0716-0943-9_2
    Gene delivery using invasive bacteria as vectors is a robust method that is feasible for plasmid and artificial chromosome DNA construct delivery to human cells presenting β1 integrin receptors. This technique is relatively underutilized owing to the inefficiency of gene transfer to targeted cell populations. Bacterial vectors must successfully adhere to the cell membrane, internalize into the cytoplasm, undergo lysis, and deliver DNA to the nucleus. There are limited studies on the use of exogenous reagents to improve the efficiency of bacteria-mediated gene delivery to mammalian cells. In this chapter, we describe how cationic lipids, conventionally used for DNA and protein transfection, as well as antimicrobial compounds, can be used to synergistically enhance the adherence of invasive bacterial vectors to the cell membrane and improve their predisposition to internalize into the cytoplasm to deliver DNA. Using simple combinatorial methods, functional DNA transfer can be improved by up to four-fold of invaded cell populations. These methods are easy to perform and are likely to be applicable for other bacterial vectors including Listeria and Salmonella.
    Matched MeSH terms: Genetic Vectors/genetics*
  10. Fulltext A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system
    Kalidasan V, Ng WH, Ishola OA, Ravichantar N, Tan JJ, Das KT
    Sci Rep, 2021 Sep 28;11(1):19265.
    PMID: 34584147 DOI: 10.1038/s41598-021-98657-7
    Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.
    Matched MeSH terms: Genetic Vectors/genetics*
  11. Construction of a novel inducible expression vector for Lactococcus lactis M4 and Lactobacillus plantarum Pa21
    Maidin MS, Song AA, Jalilsood T, Sieo CC, Yusoff K, Rahim RA
    Plasmid, 2014 Jul;74:32-8.
    PMID: 24879963 DOI: 10.1016/j.plasmid.2014.05.003
    A vector that drives the expression of the reporter gusA gene in both Lactobacillus plantarum and Lactococcus lactis was constructed in this study. This vector contained a newly characterized heat shock promoter (Phsp), amplified from an Enterococcus faecium plasmid, pAR6. Functionality and characterization of this promoter was initially performed by cloning Phsp into pNZ8008, a commercial lactococcal plasmid used for screening of putative promoters which utilizes gusA as a reporter. It was observed that Phsp was induced under heat, salinity and alkaline stresses or a combination of all three stresses. The newly characterized Phsp promoter was then used to construct a novel Lactobacillus vector, pAR1801 and its ability to express the gusA under stress-induced conditions was reproducible in both Lb. plantarum Pa21 and L. lactis M4 hosts.
    Matched MeSH terms: Genetic Vectors/genetics*
  12. Controlled expression of cholera toxin B subunit from Vibrio cholerae in Escherichia coli
    Haryanti T, Mariana NS, Latifah SY, Yusoff K, Raha AR
    Pak J Biol Sci, 2008 Jul 01;11(13):1718-22.
    PMID: 18819625
    The ctxB gene, the causative agent of cholera epidemic was successfully cloned from V. cholerae in E. coli. The insertion of the gene was confirmed by PCR as well as restriction digestion analyses. The sequencing results for the gene confirmed that the insert was in the correct orientation and in-frame with the P(BAD) promoter and it showed that the gene was 99% homologous to the published ctxB sequence. The CTB protein was successfully expressed in E. coli using the pBAD/His vector system. The expected protein of approximately 14 kDa was detected by SDS-PAGE and Western blot. The use of pBAD/His vector to express the cholera toxin gene in E. coli would facilitate future study of toxin gene products.
    Matched MeSH terms: Genetic Vectors/genetics
  13. Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos
    Ariffin N, Abdullah R, Rashdan Muad M, Lourdes J, Emran NA, Ismail MR, et al.
    Plasmid, 2011 Sep;66(3):136-43.
    PMID: 21827784 DOI: 10.1016/j.plasmid.2011.07.002
    Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.
    Matched MeSH terms: Genetic Vectors/genetics*
  14. RNA interference: a promising biotechnological approach to combat plant pathogens, mechanism and future prospects
    Ali A, Shahbaz M, Ölmez F, Fatima N, Umar UUD, Ali MA, et al.
    World J Microbiol Biotechnol, 2024 Oct 03;40(11):339.
    PMID: 39358476 DOI: 10.1007/s11274-024-04143-3
    Plant pathogens and other biological pests represent significant obstacles to crop Protection worldwide. Even though there are many effective conventional methods for controlling plant diseases, new methods that are also effective, environmentally safe, and cost-effective are required. While plant breeding has traditionally been used to manipulate the plant genome to develop resistant cultivars for controlling plant diseases, the emergence of genetic engineering has introduced a completely new approach to render plants resistant to bacteria, nematodes, fungi, and viruses. The RNA interference (RNAi) approach has recently emerged as a potentially useful tool for mitigating the inherent risks associated with the development of conventional transgenics. These risks include the use of specific transgenes, gene control sequences, or marker genes. Utilizing RNAi to silence certain genes is a promising solution to this dilemma as disease-resistant transgenic plants can be generated within a legislative structure. Recent investigations have shown that using target double stranded RNAs via an effective vector system can produce significant silencing effects. Both dsRNA-containing crop sprays and transgenic plants carrying RNAi vectors have proven effective in controlling plant diseases that threaten commercially significant crop species. This article discusses the methods and applications of the most recent RNAi technology for reducing plant diseases to ensure sustainable agricultural yields.
    Matched MeSH terms: Genetic Vectors/genetics
  15. Engineered AAV capsids mediate transduction of murine neurofibroma and sciatic nerve
    Abou Haidar E, Prabhakar S, Cheah PS, Hanlon KS, Espinoza P, Crain AV, et al.
    Gene Ther, 2025 Jul;32(4):385-397.
    PMID: 40494929 DOI: 10.1038/s41434-025-00542-9
    Genetic diseases such as Neurofibromatosis type 1 (NF1) and Charcot-Marie Tooth disease involve Schwann cells (SCs) associated with peripheral nerves. Gene therapy using adeno-associated virus (AAV) vector mediated gene delivery is a promising strategy to treat these diseases. However, AAV-mediated transduction of SCs in vivo after intravascular delivery is relatively inefficient, with a lack of extensive characterization of different capsids to date. Here, we performed an in vivo selection with an AAV9 capsid peptide display library in a mouse model of NF1. We chose one capsid variant, AAV-SC3, which was present in NF1 nerves for comparison to two benchmark capsids after systemic injection. AAV-SC3 significantly outperformed one of the two benchmark capsids at levels of transgene mRNA in the neurofibroma. Immunofluorescence microscopy revealed transgene expressing Sox10-positive SCs throughout the neurofibroma with AAV-SC3 injection. Next, we performed a pooled screen with four of the top capsids from our initial selection and AAV9 and identified one capsid, AAV-SC4, with enhanced biodistribution to and transduction of normal sciatic nerve in mice. This capsid displayed a peptide with a known laminin-binding motif, which may provide a conduit for future laminin-targeting strategies. Our results provide a baseline for future AAV-based gene therapies developed for NF1 or other diseases that affect SCs.
    Matched MeSH terms: Genetic Vectors/genetics
  16. Fulltext Cassette hybridization for vector assembly application in antibody chain shuffling
    Lai JY, Loh Q, Choong YS, Lim TS
    Biotechniques, 2018 11;65(5):269-274.
    PMID: 30394125 DOI: 10.2144/btn-2018-0031
    Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.
    Matched MeSH terms: Genetic Vectors/genetics*
  17. Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line
    Lazarev VN, Parfenova TM, Gularyan SK, Misyurina OY, Akopian TA, Govorun VM
    Int J Antimicrob Agents, 2002 Feb;19(2):133-7.
    PMID: 11850166
    As the number of pathogenic microbial strains resistant to different antibiotics increases, amphipathic peptides with antimicrobial activity are promising agents for the therapy of infectious diseases. This work deals with the effect of an amphipathic antimicrobial peptide, melittin, expressed within recombinant plasmid vectors, on infection with urogenital pathogens Chlamydia trachomatis and Mycoplasma hominis in HeLa cell culture. Recombinant plasmid constructs with the melittin gene under the control of the tetracycline-responsive promoter of human cytomegalovirus were obtained. We showed inhibition of C. trachomatis and M. hominis infection after the introduction of recombinant plasmid vectors expressing the melittin gene into the infected cell culture.
    Matched MeSH terms: Genetic Vectors/genetics
  18. Fulltext A comparative study on the expression, purification and functional characterization of human adiponectin in Pichia pastoris and Escherichia coli
    Rothan HA, Teh SH, Haron K, Mohamed Z
    Int J Mol Sci, 2012;13(3):3549-62.
    PMID: 22489167 DOI: 10.3390/ijms13033549
    Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.
    Matched MeSH terms: Genetic Vectors/genetics
  19. IRES-incorporated lactococcal bicistronic vector for target gene expression in a eukaryotic system
    Abdul Mutalib NE, Mat Isa N, Alitheen NB, Song AA, Rahim RA
    Plasmid, 2014 May;73:26-33.
    PMID: 24780699 DOI: 10.1016/j.plasmid.2014.04.003
    Plasmid DNAs isolated from lactic acid bacteria (LAB) such as Lactococcus lactis (L. lactis) has been gaining more interests for its positive prospects in genetic engineering-related applications. In this study, the lactococcal plasmid, pNZ8048 was modified so as to be able to express multiple genes in the eukaryotic system. Therefore, a cassette containing an internal ribosome entry site (IRES) was cloned between VP2 gene of a very virulent infectious bursal disease (vvIBDV) UPM 04190 of Malaysian local isolates and the reporter gene, green fluorescent protein (GFP) into pNZ:CA, a newly constructed derivative of pNZ8048 harboring the cytomegalovirus promoter (Pcmv) and polyadenylation signal. The new bicistronic vector, denoted as pNZ:vig was subjected to in vitro transcription/translation system followed by SDS-PAGE and Western blot analysis to rapidly verify its functionality. Immunoblotting profiles showed the presence of 49 and 29kDa bands that corresponds to the sizes of the VP2 and GFP proteins respectively. This preliminary result shows that the newly constructed lactococcal bicistronic vector can co-express multiple genes in a eukaryotic system via the IRES element thus suggesting its feasibility to be used for transfection of in vitro cell cultures and vaccine delivery.
    Matched MeSH terms: Genetic Vectors/genetics*
  20. Fulltext Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli
    Mualif SA, Teow SY, Omar TC, Chew YW, Yusoff NM, Ali SA
    PLoS One, 2015;10(7):e0130446.
    PMID: 26147991 DOI: 10.1371/journal.pone.0130446
    Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.
    Matched MeSH terms: Genetic Vectors/genetics*
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