Displaying publications 1 - 20 of 22 in total

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  1. Yew CW, Kumar SV
    Mol Biol Rep, 2012 Feb;39(2):1783-90.
    PMID: 21625851 DOI: 10.1007/s11033-011-0919-7
    MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21-24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.
    Matched MeSH terms: Cloning, Molecular/methods*
  2. Rahman RN, Leow TC, Basri M, Salleh AB
    Protein Expr. Purif., 2005 Apr;40(2):411-6.
    PMID: 15766884
    The extracellular production of T1 lipase was performed by co-expression of pJL3 vector encoding bacteriocin release protein in prokaryotic system. Secretory expression was optimized by considering several parameters, including host strains, inducer (IPTG) concentration, media, induction at A(600 nm), temperature, and time of induction. Among the host strains tested, Origami B excreted out 18,100 U/ml of lipase activity into culture medium when induced with 50 microM IPTG for 12 h. The Origami B harboring recombinant plasmid pGEX/T1S and pJL3 vector was chosen for further study. IPTG at 0.05 mM, YT medium, induction at A(600 nm) of 1.25, 30 degrees C, and 32 h of induction time were best condition for T1 lipase secretion with Origami B as a host.
    Matched MeSH terms: Cloning, Molecular/methods*
  3. Duff-Farrier CRA, Mbanzibwa DR, Nanyiti S, Bunawan H, Pablo-Rodriguez JL, Tomlinson KR, et al.
    Mol Biotechnol, 2019 Feb;61(2):93-101.
    PMID: 30484144 DOI: 10.1007/s12033-018-0139-7
    Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.
    Matched MeSH terms: Cloning, Molecular/methods*
  4. Ch'ng ACW, Hamidon NHB, Konthur Z, Lim TS
    Methods Mol Biol, 2018;1701:301-319.
    PMID: 29116512 DOI: 10.1007/978-1-4939-7447-4_16
    The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning experiments. There are both manual and semi-automated panning selections using polystyrene plastic, magnetic beads, and nitrocellulose as the immobilizing solid surface. Magnetic nanoparticles allow for improved antigen binding due to their large surface area. The Kingfisher Flex magnetic particle processing system was originally designed to aid in RNA, DNA, and protein extraction using magnetic beads. However, the system can be programmed for antibody phage display panning. The automation allows for a reduction in human error and improves reproducibility in between selections with the preprogrammed movements. The system requires minimum human intervention to operate; however, human intervention is needed for post-panning steps like phage rescue. In addition, polyclonal and monoclonal ELISA can be performed using the semi-automated platform to evaluate the selected antibody clones. This chapter will summarize the suggested protocol from the panning stage till the monoclonal ELISA evaluation. Other than this, important notes on the possible optimization and troubleshooting are also included at the end of this chapter.
    Matched MeSH terms: Cloning, Molecular/methods*
  5. Chin CF, Choong YS, Lim TS
    Methods Mol Biol, 2018;1701:285-299.
    PMID: 29116511 DOI: 10.1007/978-1-4939-7447-4_15
    Antibody phage display has been widely established as the method of choice to generate monoclonal antibodies with various efficacies post hybridoma technology. This technique is a popular method which takes precedence over ease of methodology, time- and cost-savings with comparable outcomes to conventional methods. Phage display technology manipulates the genome of M13 bacteriophage to display large diverse collection of antibodies that is capable of binding to various targets (nucleic acids, peptides, proteins, and carbohydrates). This subsequently leads to the discovery of target-related antibody binders. There have been several different approaches adapted for antibody phage display over the years. This chapter focuses on the semi-automated phage display antibody biopanning method utilizing the MSIA™ streptavidin D.A.R.T's® system. The system employs the use of electronic multichannel pipettes with predefined programs to carry out the panning process. The method should also be adaptable to larger liquid handling instrumentations for higher throughput.
    Matched MeSH terms: Cloning, Molecular/methods*
  6. Hashim NH, Bharudin I, Nguong DL, Higa S, Bakar FD, Nathan S, et al.
    Extremophiles, 2013 Jan;17(1):63-73.
    PMID: 23132550 DOI: 10.1007/s00792-012-0494-4
    The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.
    Matched MeSH terms: Cloning, Molecular/methods
  7. 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: Cloning, Molecular/methods
  8. Pang SL, Ho KL, Waterman J, Teh AH, Chew FT, Ng CL
    Acta Crystallogr F Struct Biol Commun, 2015 Nov;71(Pt 11):1396-400.
    PMID: 26527267 DOI: 10.1107/S2053230X1501818X
    Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
    Matched MeSH terms: Cloning, Molecular/methods*
  9. 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: Cloning, Molecular/methods
  10. Moriya S, Khel NB, Parhar IS
    Neuroscience, 2015 May 21;294:109-15.
    PMID: 25772790 DOI: 10.1016/j.neuroscience.2015.03.012
    Serotonin (5-HT) is a key regulator of mood and sexual behaviors. 5-HT reuptake inhibitors have been used as antidepressants. Really interesting new gene (RING) finger proteins have been associated with 5-HT regulation but their role remains largely unknown. Some RING finger proteins are involved in the serotonergic system, therefore, we speculate that the gene expression of RING finger protein38 (rnf38) is regulated by the serotonergic system. In the present study, we aimed to identify the full length sequence of medaka (Oryzias latipes) rnf38 mRNA and investigate its association with the serotonergic system using an antidepressant, citalopram (CIT). We identified the full length rnf38 cDNA, which consisted of 2726 nucleotides spanning 12 exons and the deduced protein sequence consisting of 518 amino acid residues including a RING finger domain, a KIT motif and a coiled-coil domain. Medaka exposed to 10(-7)M of CIT showed anxiety-like behavior. The expressions of 5-HT-related genes, pet1, solute carrier family 6, member 4A (slc6a4) and tryptophan hydroxylase (tph2) were significantly low (P<0.05) in the hindbrain. On the other hand, rnf38 gene was significantly high (P<0.05) in the telencephalon and the hypothalamus. This shows that 5-HT synthesis and transport in the hindbrain is suppressed by CIT, which induces rnf38 gene expression in the forebrain where 5-HT neurons project. Thus, the expression of rnf38 is negatively regulated by the serotonergic system.
    Matched MeSH terms: Cloning, Molecular/methods
  11. 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: Cloning, Molecular/methods
  12. Lai JY, Klatt S, Lim TS
    Crit Rev Biotechnol, 2019 May;39(3):380-394.
    PMID: 30720351 DOI: 10.1080/07388551.2019.1566206
    Through the discovery of monoclonal antibody (mAb) technology, profound successes in medical treatment against a wide range of diseases have been achieved. This has led antibodies to emerge as a new class of biodrugs. As the "rising star" in the pharmaceutical market, extensive research and development in antibody production has been carried out in various expression systems including bacteria, insects, plants, yeasts, and mammalian cell lines. The major benefit of eukaryotic expression systems is the ability to carry out posttranslational modifications of the antibody. Glycosylation of therapeutic antibodies is one of these important modifications, due to its influence on antibody structure, stability, serum half-life, and complement recruitment. In recent years, the protozoan parasite Leishmania tarentolae has been introduced as a new eukaryotic expression system. L. tarentolae is rich in glycoproteins with oligosaccharide structures that are very similar to humans. Therefore, it is touted as a potential alternative to mammalian expression systems for therapeutic antibody production. Here, we present a comparative review on the features of the L. tarentolae expression system with other expression platforms such as bacteria, insect cells, yeasts, transgenic plants, and mammalian cells with a focus on mAb production.
    Matched MeSH terms: Cloning, Molecular/methods*
  13. Garba L, Ali MS, Oslan SN, Rahman RN
    Mol Biotechnol, 2016 Nov;58(11):718-728.
    PMID: 27629791
    Fatty acid desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated fatty acyl-chains to produce unsaturated fatty acids. A gene coding for a putative Δ9-fatty acid desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-fatty acid desaturase-like protein activity and increased overall levels of cellular unsaturated fatty acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic acid to oleic acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-fatty acid desaturase protein actively expressed in E. coli under the influence of temperature and an inducer.
    Matched MeSH terms: Cloning, Molecular/methods*
  14. 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: Cloning, Molecular/methods
  15. Yu G, Hatta A, Periyannan S, Lagudah E, Wulff BBH
    Methods Mol Biol, 2017;1659:207-213.
    PMID: 28856653 DOI: 10.1007/978-1-4939-7249-4_18
    DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, β-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform-isoamyl alcohol (24:1) or 6 M ammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).
    Matched MeSH terms: Cloning, Molecular/methods
  16. Steuernagel B, Periyannan SK, Hernández-Pinzón I, Witek K, Rouse MN, Yu G, et al.
    Nat Biotechnol, 2016 Jun;34(6):652-5.
    PMID: 27111722 DOI: 10.1038/nbt.3543
    Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.
    Matched MeSH terms: Cloning, Molecular/methods*
  17. Zainal Z, Sajari R, Ismail I
    J. Biochem. Mol. Biol. Biophys., 2002 Dec;6(6):415-9.
    PMID: 14972797
    Ornithine decarboxylase (ODC) is an enzyme of one of the two pathways of putrescine biosynthesis in plants. The genes encoding ODC have previously been cloned from Datura stramonium and human. Using differential screening, we isolated ODC cDNA clone from a cDNA library of ripening Capsicum annuum fruit. The cDNA clone designated CUKM10 contains an insert of 1523 bp. The longest open reading frame potentially encodes a peptide of 345 amino acids with an estimated molecular mass of 47 kDa and exhibit striking similarity to other ODCs. Expression analysis showed that the capODC hybridised to a single transcript with a size of 1.7 kb. The capODC transcript was first observed in early ripening and increased steadily until it reached fully ripening stage. From the observation it is suggested that capODC is developmentally regulated especially during later stage of ripening.
    Matched MeSH terms: Cloning, Molecular/methods*
  18. Sunderasan E, Bahari A, Arif SA, Zainal Z, Hamilton RG, Yeang HY
    Clin Exp Allergy, 2005 Nov;35(11):1490-5.
    PMID: 16297147 DOI: 10.1111/j.1365-2222.2005.02371.x
    BACKGROUND:
    Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information.

    OBJECTIVE:
    We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding.

    METHODS:
    The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients.

    RESULTS:
    The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue.

    CONCLUSION:
    The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.
    Matched MeSH terms: Cloning, Molecular/methods*
  19. Karim KMR, Husaini A, Sing NN, Tasnim T, Mohd Sinang F, Hussain H, et al.
    Protein Expr. Purif., 2019 12;164:105462.
    PMID: 31351992 DOI: 10.1016/j.pep.2019.105462
    The Aspergillus flavus NSH9 gene, encoding a pH and thermostable glucoamylase with a starch binding domain (SBD), was expressed in Pichia pastoris to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp), and cDNA (1839 bp) encode a 612 amino acid protein most similar to glucoamylase from Aspergillus oryzae RIB40; the first 19 amino acids are presumed to be a signal peptide for secretion, and the SBD is at the C-terminal. The cDNA was successfully secreted by Pichia at 8.23 U mL-1, and the rGA2 was found to be: a 80 kDa monomer, stable from pH 3.0-9.0, with optimum catalytic activity at pH 5.0, active at temperatures up to 80°C (rGA2 retained 58% of its activity after 60 min of incubation at 70°C), and metal ions such as Na+, K+, Ca++ and Mg++ enhanced rGA2 enzyme activity. The starch degrading ability of rGA2 was also observed on raw sago starch and where prolonged incubation generated larger, deeper, holes on the starch granules, indicating rGA2 is an excellent candidate for industrial starch processing applications.
    Matched MeSH terms: Cloning, Molecular/methods
  20. Karim KM, Husaini A, Hossain MA, Sing NN, Mohd Sinang F, Hussain MH, et al.
    Biomed Res Int, 2016;2016:5962028.
    PMID: 27504454 DOI: 10.1155/2016/5962028
    A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.
    Matched MeSH terms: Cloning, Molecular/methods
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