Journal/Magazine Articles

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This collection contains original research articles, review articles and case reports published in local and international peer reviewed journals by the staff members of the Faculty of Medicine

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Subject: search.filters.subject.Recombinant Proteins

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    Heterologous expression, chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata
    (Asian Pacific Tropical Medicine Press , China, 2014) Rodrigo, W.W.P.; Dassanayake, R.S.; Karunanayake, E.H.; Gunawardene, Y.I.N.S.; Weerasena, O.V.D.S.J.
    OBJECTIVE: To clone, express and purify a putative parasitic nematode specific protein of Setaria digitata (S. digitata), filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to be used for structural and functional analyses. METHODS: To characterize uncharacterized gene of S. digitata (SDUG), the herterologous expression of SDUG was carried out in the pET [cloned into pET45b(+)] expression system initially and co-expression of SDUG using chaperone plasmids pG-KJE8, pGro 7, pKJE7, pG-Tf2 and pTf16 containing chaperone proteins of dnaK-dnaJ-grpE-groES-gro-E, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig respectively, was carried out subsequently. RESULTS: Expression of SDUG was seen when Escherichia coli strain BL21(DE3) is used, while concentrating protein largely into the insoluble fraction. The co-expression of SDUG using chaperone plasmid mediated system indicated a significant increase of the protein in the soluble fraction. Of the chaperon plasmid sets, the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of 2 mg/mL L-arabinose and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28 °C. Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography. SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of ∼24 kDa. CONCLUSIONS: SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.
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    Characterization of a novel cellular retinoic acid/retinol binding protein from shrimp: expression of the recombinant protein for immunohistochemical detection and binding assay
    (Elsevier/North-Holland, 2002) Gu, P.L.; Gunawardene, Y.I.N.S.; Chow, B.C.; He, J.G.; Chan, S.M.
    Members of the cellular retinoic acid (CRABP) and retinol binding (CRBP) proteins family are involved in the metabolic pathways of retinoic acid (RA) and retinal respectively. The objective of this study is to determine whether such proteins are present in crustaceans. We report here the cloning and isolation of a novel complementary DNA (cDNA) that showed characteristics of the CRABP/CRBP from the ovary and eyestalk of the shrimp. The cDNA is 0.9 Kb in size and the deduced shrimp protein is encoded for a protein of 14 kDa. Although it shows high amino acids sequence similarity to both the vertebrate and invertebrate CRABP, some conserved amino acids identified in other CRABPs were not found in MeCRABP. MeCRABP is expressed in the ovary, eyestalk, testis, epidermis and early larvae. The presence of MeCRABP in early larval stages suggests that the protein may be involved in the early larval development. Recombinant MeCRABP was produced and used to generate a polyclonal antibody. In theimmunohistochemical detection study, anti-rCRABP antibody recognized the presence of CRABP in several cell types of the eyestalk as well as the smaller oocytes of the ovary. Although MeCRABP messenger RNA transcripts can be detected in the ovary throughout the ovarian maturation period, CRABP was detected only in the primary oocytes of the ovary. The results suggest that CRABP transcripts in the mature ovary are not translated and may be supplied to the oocyte as maternal messages. The binding property of the recombinant MeCRABP was also tested by a fluorometeric method. The result indicates that rMeCRABP binds to both RA and retinal with similar affinity. This study represents the first cloning andcharacterization of a cDNA that belongs to a member of retinoid/fatty acid binding protein family in crustaceans.