Displaying publications 1 - 20 of 71 in total

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  1. Designing and evaluation of an antibody-targeted chimeric recombinant vaccine encoding Shigella flexneri outer membrane antigens
    Kazi A, Hisyam Ismail CMK, Anthony AA, Chuah C, Leow CH, Lim BH, et al.
    Infect Genet Evol, 2020 06;80:104176.
    PMID: 31923724 DOI: 10.1016/j.meegid.2020.104176
    Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection.
    Matched MeSH terms: Recombinant Fusion Proteins
  2. Fulltext Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells
    Munisvaradass R, Kumar S, Govindasamy C, Alnumair KS, Mok PL
    Int J Mol Sci, 2017 Sep 08;18(9).
    PMID: 28885562 DOI: 10.3390/ijms18091797
    Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non-transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours.
    Matched MeSH terms: Recombinant Fusion Proteins
  3. PAK5 mediates cell: cell adhesion integrity via interaction with E-cadherin in bladder cancer cells
    Ismail AF, Oskay Halacli S, Babteen N, De Piano M, Martin TA, Jiang WG, et al.
    Biochem. J., 2017 Mar 24;474(8):1333-1346.
    PMID: 28232500 DOI: 10.1042/BCJ20160875
    Urothelial bladder cancer is a major cause of morbidity and mortality worldwide, causing an estimated 150 000 deaths per year. Whilst non-muscle-invasive bladder tumours can be effectively treated, with high survival rates, many tumours recur, and some will progress to muscle-invasive disease with a much poorer long-term prognosis. Thus, there is a pressing need to understand the molecular transitions occurring within the progression of bladder cancer to an invasive disease. Tumour invasion is often associated with a down-regulation of E-cadherin expression concomitant with a suppression of cell:cell junctions, and decreased levels of E-cadherin expression have been reported in higher grade urothelial bladder tumours. We find that expression of E-cadherin in a panel of bladder cancer cell lines correlated with the presence of cell:cell junctions and the level of PAK5 expression. Interestingly, exogenous PAK5 has recently been described to be associated with cell:cell junctions and we now find that endogenous PAK5 is localised to cell junctions and interacts with an E-cadherin complex. Moreover, depletion of PAK5 expression significantly reduced junctional integrity. These data suggest a role for PAK5 in maintaining junctional stability and we find that, in both our own patient samples and a commercially available dataset, PAK5mRNA levels are reduced in human bladder cancer compared with normal controls. Taken together, the present study proposes that PAK5 expression levels could be used as a novel prognostic marker for bladder cancer progression.
    Matched MeSH terms: Recombinant Fusion Proteins/metabolism
  4. Immunological characterization of rice tungro spherical virus coat proteins and differentiation of isolates from the Philippines and India
    Druka A, Burns T, Zhang S, Hull R
    J Gen Virol, 1996 Aug;77 ( Pt 8):1975-83.
    PMID: 8760450
    Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.
    Matched MeSH terms: Recombinant Fusion Proteins
  5. Gonadotropin-inhibitory hormone promoter-driven enhanced green fluorescent protein expression decreases during aging in female rats
    Soga T, Kitahashi T, Clarke IJ, Parhar IS
    Endocrinology, 2014 May;155(5):1944-55.
    PMID: 24605826 DOI: 10.1210/en.2013-1786
    Gonadotropin-inhibitory hormone (GnIH) neurons project to GnRH neurons to negatively regulate reproductive function. To fully explore the projections of the GnIH neurons, we created transgenic rats carrying an enhanced green fluorescent protein (EGFP) tagged to the GnIH promoter. With these animals, we show that EGFP-GnIH neurons are localized mainly in the dorsomedial hypothalamic nucleus (DMN) and project to the hypothalamus, telencephalon, and diencephalic thalamus, which parallels and confirms immunocytochemical and gene expression studies. We observed an age-related reduction in c-Fos-positive GnIH cell numbers in female rats. Furthermore, GnIH fiber appositions to GnRH neurons in the preoptic area were lessened in middle-aged females (70 weeks old) compared with their younger counterparts (9-12 weeks old). The fiber density in other brain areas was also reduced in middle-aged female rats. The expression of estrogen and progesterone receptors mRNA in subsets of EGFP-GnIH neurons was shown in laser-dissected single EGFP-GnIH neurons. We then examined estradiol-17β and progesterone regulation of GnIH neurons, using c-Fos presence as a marker. Estradiol-17β treatment reduced c-Fos labeling in EGFP-GnIH neurons in the DMN of young ovariectomized adult females but had no effect in middle-aged females. Progesterone had no effect on the number of GnIH cells positive for c-Fos. We conclude that there is an age-related decline in GnIH neuron number and GnIH inputs to GnRH neurons. We also conclude that the response of GnIH neurons to estrogen diminishes with reproductive aging.
    Matched MeSH terms: Recombinant Fusion Proteins/metabolism
  6. Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus
    Thanh T, Chi VT, Abdullah MP, Omar H, Noroozi M, Napis S
    Mol Biol Rep, 2011 Nov;38(8):5297-305.
    PMID: 21287365 DOI: 10.1007/s11033-011-0679-4
    An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.
    Matched MeSH terms: Recombinant Fusion Proteins/metabolism
  7. Fulltext Immunogenicity of recombinant Mycobacterium bovis bacille Calmette-Guèrin clones expressing T and B cell epitopes of Mycobacterium tuberculosis antigens
    Mohamud R, Azlan M, Yero D, Alvarez N, Sarmiento ME, Acosta A, et al.
    BMC Immunol, 2013;14 Suppl 1:S5.
    PMID: 23458635 DOI: 10.1186/1471-2172-14-S1-S5
    Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4⁺ and CD8⁺ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.
    Matched MeSH terms: Recombinant Fusion Proteins/immunology
  8. 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.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  9. Fulltext AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation
    Cullen JK, Abdul Murad N, Yeo A, McKenzie M, Ward M, Chong KL, et al.
    PLoS One, 2016;11(2):e0148213.
    PMID: 26866375 DOI: 10.1371/journal.pone.0148213
    Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients.
    Matched MeSH terms: Recombinant Fusion Proteins/chemistry
  10. Current updates on pharmacological roles of glucagon-like peptide 1 in obesity
    Verma RK, Sriramaneni R, Pandey M, Chaudhury H, Gorain B, Gupta G
    Panminerva Med, 2018 Dec;60(4):224-225.
    PMID: 29856185 DOI: 10.23736/S0031-0808.18.03479-1
    Matched MeSH terms: Recombinant Fusion Proteins/therapeutic use
  11. Inhibitory effects of a peptide-fusion protein (Latarcin-PAP1-Thanatin) against chikungunya virus
    Rothan HA, Bahrani H, Shankar EM, Rahman NA, Yusof R
    Antiviral Res, 2014 Aug;108:173-80.
    PMID: 24929084 DOI: 10.1016/j.antiviral.2014.05.019
    Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/pharmacology; Recombinant Fusion Proteins/therapeutic use
  12. Cell surface display system for Lactococcus lactis: a novel development for oral vaccine
    Raha AR, Varma NR, Yusoff K, Ross E, Foo HL
    Appl Microbiol Biotechnol, 2005 Jul;68(1):75-81.
    PMID: 15635459
    The food-grade Lactococcus lactis is a potential vector to be used as a live vehicle for the delivery of heterologous proteins for vaccine and pharmaceutical purposes. We constructed a plasmid vector pSVac that harbors a 255-bp single-repeat sequence of the cell wall-binding protein region of the AcmA protein. The recombinant plasmid was transformed into Escherichia coli and expression of the gene fragment was driven by the T7 promoter of the plasmid. SDS-PAGE showed the presence of the putative AcmA' fragment and this was confirmed by Western blot analysis. The protein was isolated and purified using a His-tag affinity column. When mixed with a culture of L. lactis MG1363, ELISA and immunofluorescence assays showed that the cell wall-binding fragment was anchored onto the outer surface of the bacteria. This indicated that the AcmA' repeat unit retained the active site for binding onto the cell wall surface of the L. lactis cells. Stability assays showed that the fusion proteins (AcmA/A1, AcmA/A3) were stably docked onto the surface for at least 5 days. The AcmA' fragment was also shown to be able to strongly bind onto the cell surface of naturally occurring lactococcal strains and Lactobacillus and, with less strength, the cell surface of Bacillus sphericus. The new system designed for cell surface display of recombinant proteins on L. lactis was evaluated for the expression and display of A1 and A3 regions of the VP1 protein of enterovirus 71 (EV71). The A1 and A3 regions of the VP1 protein of EV71 were cloned upstream to the cell wall-binding domains of AcmA protein and successfully expressed as AcmA/A1 and AcmA/A3. Whole-cell ELISA showed the successful display of VP1 protein epitopes of EV71 on the surface of L. lactis. The success of the anchoring system developed in this study for docking the A1 and A3 epitopes of VP1 onto the surface of L. lactis cells opens up the possibilities of peptide and protein display for not only Lactococcus but also for other gram-positive bacteria. This novel way of displaying epitopes on the cell surface of L. lactis and other related organisms should be very useful in the delivery of vaccines and other useful proteins.
    Matched MeSH terms: Recombinant Fusion Proteins
  13. Fulltext Inhibition of SARS-CoV-2 3CL protease by the anti-viral chimeric protein RetroMAD1
    Chan LC, Mat Yassim AS, Ahmad Fuaad AAH, Leow TC, Sabri S, Radin Yahaya RS, et al.
    Sci Rep, 2023 Nov 17;13(1):20178.
    PMID: 37978223 DOI: 10.1038/s41598-023-47511-z
    COVID-19 results from SARS-CoV-2, which mutates frequently, challenging current treatments. Therefore, it is critical to develop new therapeutic drugs against this disease. This study explores the interaction between SARS-CoV-2 3CLpro and RetroMAD1, a well-characterized coronavirus protein and potential drug target, using in-silico methods. The analysis through the HDOCK server showed stable complex formation with a binding energy of -12.3, the lowest among reference drugs. The RetroMAD1-3CLpro complex underwent a 100 ns molecular dynamics simulation (MDS) in an explicit solvation system, generating various trajectories, including RMSD, RMSF, hydrogen bonding, radius of gyration, and ligand binding energy. MDS results confirmed intact interactions within the RetroMAD1-3CLpro complex during simulations. In vitro experiments validated RetroMAD1's ability to inhibit 3CLpro enzyme activity and prevent SARS-CoV-2 infection in human bronchial cells. RetroMAD1 exhibited antiviral efficacy comparable to Remdesivir without cytotoxicity at effective concentrations. These results suggest RetroMAD1 as a potential drug candidate against SARS-CoV-2, warranting further in vivo and clinical studies to assess its efficiency.
    Matched MeSH terms: Recombinant Fusion Proteins
  14. Fulltext Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication
    Rothan HA, Bahrani H, Mohamed Z, Abd Rahman N, Yusof R
    PLoS One, 2014;9(4):e94561.
    PMID: 24722532 DOI: 10.1371/journal.pone.0094561
    Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.
    Matched MeSH terms: Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/pharmacology*
  15. Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin
    Nathan S, Li H, Mohamed R, Embi N
    J. Biochem. Mol. Biol. Biophys., 2002 Feb;6(1):45-53.
    PMID: 12186782
    We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.
    Matched MeSH terms: Recombinant Fusion Proteins/immunology
  16. Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs
    Jeyaseelan K, Armugam A, Lachumanan R, Tan CH, Tan NH
    Biochim. Biophys. Acta, 1998 Apr 10;1380(2):209-22.
    PMID: 9565688
    Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics
  17. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli
    Yee SF, Chu CH, Poili E, Sum MSH
    J Virol Methods, 2017 02;240:69-72.
    PMID: 27923590 DOI: 10.1016/j.jviromet.2016.12.001
    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.
    Matched MeSH terms: Recombinant Fusion Proteins/immunology
  18. Fulltext Characterisation of Drosophila Ubx CPTI000601 and hth CPTI000378 protein trap lines
    Choo SW, Beh CY, Russell S, White R
    ScientificWorldJournal, 2014;2014:191535.
    PMID: 25389534 DOI: 10.1155/2014/191535
    In Drosophila, protein trap strategies provide powerful approaches for the generation of tagged proteins expressed under endogenous control. Here, we describe expression and functional analysis to evaluate new Ubx and hth protein trap lines generated by the Cambridge Protein Trap project. Both protein traps exhibit spatial and temporal expression patterns consistent with the reported endogenous pattern in the embryo. In imaginal discs, Ubx-YFP is expressed throughout the haltere and 3rd leg imaginal discs, while Hth-YFP is expressed in the proximal regions of haltere and wing discs but not in the pouch region. The Ubx (CPTI000601) line is semilethal as a homozygote. No T3/A1 to T2 transformations were observed in the embryonic cuticle or the developing midgut. The homozygous survivors, however, exhibit a weak haltere phenotype with a few wing-like marginal bristles on the haltere capitellum. Although hth (CPTI000378) is completely lethal as a homozygote, the hth (CPTI000378) /hth (C1) genotype is viable. Using a hth deletion (Df(3R)BSC479) we show that hth (CPTI000378) /Df(3R)BSC479 adults are phenotypically normal. No transformations were observed in hth (CPTI000378), hth (CPTI000378) /hth (C1), or hth (CPTI000378) /Df(3R)BSC479 embryonic cuticles. We have successfully characterised the Ubx-YFP and Hth-YFP protein trap lines demonstrating that the tagged proteins show appropriate expression patterns and produce at least partially functional proteins.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics*; Recombinant Fusion Proteins/metabolism
  19. Fulltext Multimeric disintegrin protein polymer fusions that target tumor vasculature
    Janib SM, Gustafson JA, Minea RO, Swenson SD, Liu S, Pastuszka MK, et al.
    Biomacromolecules, 2014 Jul 14;15(7):2347-58.
    PMID: 24871936 DOI: 10.1021/bm401622y
    Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.
    Matched MeSH terms: Recombinant Fusion Proteins/pharmacology*; Recombinant Fusion Proteins/chemistry*
  20. Safety and Management of Toxicity Related to Aflibercept in Combination with Fluorouracil, Leucovorin and Irinotecan in Malaysian Patients with Metastatic Colorectal Cancer
    Yusof MM, Abdullah NM, Sharial MM, Zaatar A
    Asian Pac J Cancer Prev, 2016;17(3):973-8.
    PMID: 27039822
    BACKGROUND: Between October 2012 and February 2015, 25 patients with metastatic colorectal cancer (mCRC) (mean age, 57.0 ± 12.1 years) were granted access to aflibercept via the Aflibercept Named Patient Program at four centers.

    MATERIALS AND METHODS: Here we reported the initial experience of aflibercept / FOLFIRI in combination. We evaluated treatment-related adverse events (AEs), progression-free survival (PFS) and overall survival (OS).

    RESULTS: The majority of the patients experienced gastrointestinal toxicity (grade 1-2), with diarrhea (52%), mucositis (52%), and nausea/vomiting (20%) being largely observed. Neutropenia (16%) and febrile neutropenia (8%) were common grade 3-4 hematological events. Aflibercept-related toxicity was managed as per practice guidelines. No grade 5 event was reported. Median PFS was 6.12 months (95% CI, 4.80-7.20) and OS was 12 months (95% CI, 9.80-14.18). The partial response (PR), stable disease (SD), and progressive disease (PD) rates were 25% (95% CI: 23.4-27.0), 37.5% (95% CI: 31.6-43.3), and 37.5% (95% CI: 22.5-52.5), respectively.

    CONCLUSIONS: Aflibercept/FOLFIRI can be administered safely in a second line setting to Malaysian patients with mCRC, as the AEs experienced were generally reversible and manageable. The safety and efficacy outcomes were consistent with those observed in Western populations.

    Matched MeSH terms: Recombinant Fusion Proteins/administration & dosage; Recombinant Fusion Proteins/adverse effects
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