Displaying publications 21 - 40 of 123 in total

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  1. Fulltext Using transgenic plants and modified plant viruses for the development of treatments for human diseases
    Loh HS, Green BJ, Yusibov V
    Curr Opin Virol, 2017 10;26:81-89.
    PMID: 28800551 DOI: 10.1016/j.coviro.2017.07.019
    Production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. A major milestone for plant-based protein production for use in human health was achieved when Protalix BioTherapeutics produced taliglucerase alfa (Elelyso®) in suspension cultures of a transgenic carrot cell line for the treatment of patients with Gaucher's disease, was approved by the USA Food and Drug Administration in 2012. In this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases.
    Matched MeSH terms: Recombinant Proteins/metabolism*
  2. Expression of stable and active human DNA topoisomerase I in Pichia pastoris
    Chan MK, Lim SK, Miswan N, Chew AL, Noordin R, Khoo BY
    Protein Expr. Purif., 2018 Jan;141:52-62.
    PMID: 28893606 DOI: 10.1016/j.pep.2017.09.003
    This study described the isolation of the coding region of human topoisomerase I (TopoI) from MDA-MB-231 and the expression of multiple copy recombinant genes in four Pichia pastoris strains. First, polymerase chain reaction (PCR)-amplification of the enzyme coding region was performed. The PCR fragment was cloned into pPICZ-α-A vector and sequenced. It was then transformed into X33, GS115, SMD1168H and KM71H strains of Pichia. PCR-screening for positive clones was performed, and estimation of multiple copy integrants in each Pichia strain was carried out using agar plates containing increasing concentrations of Zeocin(®). The selected clones of multiple copy recombinant genes were then induced for TopoI expression in shaker flasks. GS115 and SMD1168 were found to be better Pichia strains to accommodate the recombinant gene for the expression of TopoI extracellularly. However, the DNA relaxation activity revealed that only the target enzyme in the culture supernatants of GS115-pPICZ-α-A-TopoI exhibited consistent enzyme activity over the cultivation time-points. Active enzyme activity was inhibited by Camptothecin. The enzyme produced can be used for in-house gel-based DNA relaxation assay development in performing high throughput screening for target-specific growth inhibitors that display similar effect as the TopoI inhibitors. These inhibitors may contribute to the improvement of the treatment of cancer patients.
    Matched MeSH terms: Recombinant Proteins/metabolism*
  3. Virus-like particles of Macrobrachium rosenbergii nodavirus produced in bacteria
    Goh ZH, Tan SG, Bhassu S, Tan WS
    J Virol Methods, 2011 Jul;175(1):74-9.
    PMID: 21536072 DOI: 10.1016/j.jviromet.2011.04.021
    Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.
    Matched MeSH terms: Recombinant Proteins/metabolism
  4. Fulltext Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library
    Leow CH, Fischer K, Leow CY, Braet K, Cheng Q, McCarthy J
    Malar J, 2018 Oct 24;17(1):383.
    PMID: 30355309 DOI: 10.1186/s12936-018-2531-y
    BACKGROUND: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.

    METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.

    RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.

    CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.

    Matched MeSH terms: Recombinant Proteins/metabolism
  5. Production of 3-Oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic Acid in the Fungus Aspergillus oryzae: A Step Towards Heterologous Production of Pyrethrins in Fungi
    Mohamed ME, Pahirulzaman KA, Lazarus CM
    Mol Biotechnol, 2016 Mar;58(3):172-8.
    PMID: 26718544 DOI: 10.1007/s12033-015-9911-0
    Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. Synthetic pyrethroids are more stable, more efficacious and cheaper, but contemporary requirements for safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation. The biosynthesis of pyrethrins involves ester linkage between an acid moiety (chrysanthemoyl or pyrethroyl, synthesised via the mevalonic acid pathway from glucose), and an alcohol (pyrethrolone). Pyrethrolone is generated from 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid, which originates from α-linolenic acid via the jasmonic acid biosynthetic cascade. The first four genes in this cascade, encoding lipoxygenase 2, allene-oxide synthase, allene-oxide cyclase 2 and 12-oxophytodienoic acid reductase 3, were amplified from an Arabidopsis thaliana cDNA library, cloned in a purpose-built fungal multigene expression vector and expressed in Aspergillus oryzae. HPLC-MS analysis of the transgenic fungus homogenate gave good evidence for the presence of 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid.
    Matched MeSH terms: Recombinant Proteins/metabolism
  6. Fulltext Sero-diagnostic evaluation of Toxoplasma gondii recombinant Rhoptry antigen 8 expressed in E. coli
    Sonaimuthu P, Fong MY, Kalyanasundaram R, Mahmud R, Lau YL
    Parasit Vectors, 2014;7:297.
    PMID: 24986686 DOI: 10.1186/1756-3305-7-297
    Toxoplasma gondii infects all warm-blooded animals, including humans. Early diagnosis and determining the infective stage are critical for effectively treating immunosuppressed individuals and pregnant women with toxoplasmosis. Among the rhoptry proteins of the parasite, Rhoptry protein 8 (ROP8), is known to be expressed during the early stages of T. gondii infection and is involved in parasitophorous vacuole formation. In this study, we have investigated the diagnostic efficacy of recombinant ROP8 (rROP8).
    Matched MeSH terms: Recombinant Proteins/metabolism*
  7. In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism
    Bakri MM, Rich AM, Cannon RD, Holmes AR
    Mol Oral Microbiol, 2015 Feb;30(1):27-38.
    PMID: 24975985 DOI: 10.1111/omi.12064
    Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.
    Matched MeSH terms: Recombinant Proteins/metabolism
  8. Proteome analysis of Escherichia coli periplasmic proteins in response to over-expression of recombinant human interferon α2b
    Heidary S, Rahim RA, Eissazadeh S, Moeini H, Chor AL, Abdullah MP
    Biotechnol Lett, 2014 Jul;36(7):1479-84.
    PMID: 24652546 DOI: 10.1007/s10529-014-1504-7
    The periplasmic proteome of recombinant E. coli cells expressing human interferon-α2b (INF-α2b) was analysed by 2D-gel electrophoresis to find the most altered proteins. Of some unique up- and down-regulated proteins in the proteome, ten were identified by MS. The majority of the proteins belonged to the ABC transporter protein family. Other affected proteins were ones involved in the regulation of transcription such as DNA-binding response regulator, stress-related proteins and ecotin. Thus, the production of INF-α2b acts as a stress on the cells and results in the induction of various transporters and stress related proteins.
    Matched MeSH terms: Recombinant Proteins/metabolism
  9. Immunological role of thiol-dependent peroxiredoxin gene in Macrobrachium rosenbergii
    Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S
    Fish Shellfish Immunol, 2012 Jul;33(1):121-9.
    PMID: 22565019 DOI: 10.1016/j.fsi.2012.04.010
    In this study, we have reported a full length of peroxiredoxin (designated MrPrdx) gene, identified from the transcriptome of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrPrdx is 940 base pairs in length, and encodes 186 amino acids. MrPrdx contains a long thioredoxin domain in the amino acid sequence between 34 and 186. The gene expressions of MrPrdx in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction. MrPrdx is highly expressed in all the other tissues of M. rosenbergii considered for analysis and the highest in gills. The expression is strongly up-regulated in gills after IHHNV infection. To understand MrPrdx functional properties, the recombinant MrPrdx protein was expressed in Escherichia coli BL21 (DE3) and purified. A peroxidise activity assay was conducted using recombinant MrPrdx protein at different concentrations. This peroxidises activity showed that the recombinant MrPrdx is a thiol-dependant protein. Additionally, this result showed that recombinant MrPrdx protein, as a secretory protein can remove H₂O₂ and protect DNA damage. This finding leads a possible way to propose the recombinant MrPrdx protein as an effective medicine for reactive oxygen species (ROS) related diseases.
    Matched MeSH terms: Recombinant Proteins/metabolism
  10. Over-expression of the X-linked inhibitor of apoptosis protein (XIAP) delays serum deprivation-induced apoptosis in CHO-K1 cells
    Liew JC, Tan WS, Alitheen NB, Chan ES, Tey BT
    J Biosci Bioeng, 2010 Sep;110(3):338-44.
    PMID: 20547346 DOI: 10.1016/j.jbiosc.2010.02.017
    Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP over-expression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells over-expressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression.
    Matched MeSH terms: Recombinant Proteins/metabolism*
  11. A preparative purification process for recombinant hepatitis B core antigen using online capture by expanded bed adsorption followed by size-exclusion chromatography
    Ho CW, Tan WS, Chong FC, Ling TC, Tey BT
    J Microbiol Biotechnol, 2009 Apr;19(4):416-23.
    PMID: 19421000
    Hepatitis B core antigen (HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus (HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.
    Matched MeSH terms: Recombinant Proteins/metabolism
  12. Characterization of secreted recombinant Toxoplasma gondii surface antigen 2 (SAG2) heterologously expressed by the yeast Pichia pastoris
    Fong MY, Lau YL, Zulqarnain M
    Biotechnol Lett, 2008 Apr;30(4):611-8.
    PMID: 18043869
    The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii.
    Matched MeSH terms: Recombinant Proteins/metabolism*
  13. Molecular characterization of tgd057, a novel gene from Toxoplasma gondii
    Wan KL, Chang TL, Ajioka JW
    J. Biochem. Mol. Biol., 2004 Jul 31;37(4):474-9.
    PMID: 15469736
    The expressed sequence tag (EST) effort in Toxoplasma gondii has generated a substantial amount of gene information. To exploit this valuable resource, we chose to study tgd057, a novel gene identified by a large number of ESTs that otherwise show no significant match to known sequences in the database. Northern analysis showed that tgd057 is transcribed in this tachyzoite. The complete cDNA sequence of tgd057 is 1169 bp in length. Sequence analysis revealed that tgd057 possibly adopts two polyadenylation sites, utilizes the fourth in-frame ATG for translation initiation, and codes for a secretory protein. The longest open reading frame for the tgd057 gene was cloned and expressed as a recombinant protein (rd57) in Escherichia coli. Western analysis revealed that serum against rd57 recognized a molecule of ~21 kDa in the tachyzoite protein extract. This suggests that the tgd057 gene is expressed in vivo in the parasite.
    Matched MeSH terms: Recombinant Proteins/metabolism
  14. Fulltext Thermal stability engineering of Glomerella cingulata cutinase
    Chin IS, Abdul Murad AM, Mahadi NM, Nathan S, Abu Bakar FD
    Protein Eng. Des. Sel., 2013 May;26(5):369-75.
    PMID: 23468570 DOI: 10.1093/protein/gzt007
    Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.
    Matched MeSH terms: Recombinant Proteins/metabolism
  15. Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates
    Tan YP, Ling TC, Yusoff K, Tan WS, Tey BT
    J Microbiol, 2005 Jun;43(3):295-300.
    PMID: 15995649
    In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
    Matched MeSH terms: Recombinant Proteins/metabolism
  16. Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents
    Joseph NM, Ho KL, Tey BT, Tan CS, Shafee N, Tan WS
    Biotechnol Prog, 2016 Jul 08;32(4):1038-45.
    PMID: 27088434 DOI: 10.1002/btpr.2279
    The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.
    Matched MeSH terms: Recombinant Proteins/metabolism
  17. Purification of BmR1 recombinant protein
    Arifin N, Basuni M, Lan CA, Yahya AR, Noordin R
    Protein J, 2010 Oct;29(7):509-15.
    PMID: 20845068 DOI: 10.1007/s10930-010-9281-1
    This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
    Matched MeSH terms: Recombinant Proteins/metabolism
  18. A new cold-adapted, organic solvent stable lipase from mesophilic Staphylococcus epidermidis AT2
    Kamarudin NH, Rahman RN, Ali MS, Leow TC, Basri M, Salleh AB
    Protein J, 2014 Jun;33(3):296-307.
    PMID: 24777627 DOI: 10.1007/s10930-014-9560-3
    The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
    Matched MeSH terms: Recombinant Proteins/metabolism
  19. Cloning, expression and characterization of sugarcane (Saccharum officinarum L.) transketolase
    Kalhori N, Nulit R, Go R
    Protein J, 2013 Oct;32(7):551-9.
    PMID: 24132392 DOI: 10.1007/s10930-013-9516-z
    Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants.
    Matched MeSH terms: Recombinant Proteins/metabolism
  20. Validation of a Burkholderia pseudomallei hypothetical protein and determination of its translational start codon using chromosomal integration of His-Tag coding sequence
    Yam H, Abdul Rahim A, Gim Luan O, Samian R, Abdul Manaf U, Mohamad S, et al.
    Protein J, 2012 Mar;31(3):246-9.
    PMID: 22354666 DOI: 10.1007/s10930-012-9398-5
    In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
    Matched MeSH terms: Recombinant Proteins/metabolism
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