Displaying publications 1 - 20 of 60 in total

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  1. Expression of recombinant human epidermal growth factor in Escherichia coli and characterization of its biological activity
    Abdull Razis AF, Ismail EN, Hambali Z, Abdullah MN, Ali AM, Mohd Lila MA
    Appl Biochem Biotechnol, 2008 Mar;144(3):249-61.
    PMID: 18556814
    Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.
    Matched MeSH terms: Recombinant Proteins/biosynthesis*
  2. The workability of Escherichia coli BL21 (DE3) and Pseudomonas putida KT2440 expression platforms with autodisplayed cellulases: a comparison
    Obeng EM, Brossette T, Ongkudon CM, Budiman C, Maas R, Jose J
    Appl Microbiol Biotechnol, 2018 Jun;102(11):4829-4841.
    PMID: 29675801 DOI: 10.1007/s00253-018-8987-4
    This article comparatively reports the workability of Escherichia coli BL21(DE3) and Pseudomonas putida KT2440 cell factories for the expression of three model autodisplayed cellulases (i.e., endoglucanase, BsCel5A; exoglucanase, CelK; β-glucosidase, BglA). The differentiation of the recombinant cells was restricted to their cell growth and enzyme expression/activity attributes. Comparatively, the recombinant E. coli showed higher cell growth rates but lower enzyme activities than the recombinant P. putida. However, the endo-, exoglucanase, and β-glucosidase on the surfaces of both cell factories showed activity over a broad range of pH (4-10) and temperature (30-100 °C). The pH and temperature optima were pH 6, 60 °C (BsCel5A); pH 6, 60-70 °C (CelK); and pH 6, 50 °C (BglA). Overall, the P. putida cell factory with autodisplayed enzymes demonstrated higher bioactivity and remarkable biochemical characteristics and thus was chosen for the saccharification of filter paper. A volumetric blend of the three cellulases with P. putida as the host yielded a ratio of 1:1:1.5 of endoglucanase, exoglucanase, and β-glucosidase, respectively, as the optimum blend composition for filter paper degradation. At an optical density (578 nm) of 50, the blend generated a maximum sugar yield of about 0.7 mg/ml (~ 0.08 U/g) from Whatman filter paper (Ø 6 mm, ~ 2.5 mg) within 24 h.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  3. Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in Escherichia coli
    Lew MH, Lim RL
    Appl Microbiol Biotechnol, 2016 Jan;100(2):661-71.
    PMID: 26411458 DOI: 10.1007/s00253-015-6953-y
    Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  4. Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches
    Packiam KAR, Ramanan RN, Ooi CW, Krishnaswamy L, Tey BT
    Appl Microbiol Biotechnol, 2020 Apr;104(8):3253-3266.
    PMID: 32076772 DOI: 10.1007/s00253-020-10454-w
    Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.
    Matched MeSH terms: Recombinant Proteins/biosynthesis*
  5. Fulltext A review of machine learning methods to predict the solubility of overexpressed recombinant proteins in Escherichia coli
    Habibi N, Mohd Hashim SZ, Norouzi A, Samian MR
    BMC Bioinformatics, 2014;15:134.
    PMID: 24885721 DOI: 10.1186/1471-2105-15-134
    Over the last 20 years in biotechnology, the production of recombinant proteins has been a crucial bioprocess in both biopharmaceutical and research arena in terms of human health, scientific impact and economic volume. Although logical strategies of genetic engineering have been established, protein overexpression is still an art. In particular, heterologous expression is often hindered by low level of production and frequent fail due to opaque reasons. The problem is accentuated because there is no generic solution available to enhance heterologous overexpression. For a given protein, the extent of its solubility can indicate the quality of its function. Over 30% of synthesized proteins are not soluble. In certain experimental circumstances, including temperature, expression host, etc., protein solubility is a feature eventually defined by its sequence. Until now, numerous methods based on machine learning are proposed to predict the solubility of protein merely from its amino acid sequence. In spite of the 20 years of research on the matter, no comprehensive review is available on the published methods.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  6. Fulltext Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli
    Teow SY, Mualif SA, Omar TC, Wei CY, Yusoff NM, Ali SA
    BMC Biotechnol, 2013;13:107.
    PMID: 24304876 DOI: 10.1186/1472-6750-13-107
    HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-1 CA.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  7. Fulltext Microtiter miniature shaken bioreactor system as a scale-down model for process development of production of therapeutic alpha-interferon2b by recombinant Escherichia coli
    Tan JS, Abbasiliasi S, Kadkhodaei S, Tam YJ, Tang TK, Lee YY, et al.
    BMC Microbiol, 2018 01 04;18(1):3.
    PMID: 29439680 DOI: 10.1186/s12866-017-1145-9
    BACKGROUND: Demand for high-throughput bioprocessing has dramatically increased especially in the biopharmaceutical industry because the technologies are of vital importance to process optimization and media development. This can be efficiently boosted by using microtiter plate (MTP) cultivation setup embedded into an automated liquid-handling system. The objective of this study was to establish an automated microscale method for upstream and downstream bioprocessing of α-IFN2b production by recombinant Escherichia coli. The extraction performance of α-IFN2b by osmotic shock using two different systems, automated microscale platform and manual extraction in MTP was compared.

    RESULTS: The amount of α-IFN2b extracted using automated microscale platform (49.2 μg/L) was comparable to manual osmotic shock method (48.8 μg/L), but the standard deviation was 2 times lower as compared to manual osmotic shock method. Fermentation parameters in MTP involving inoculum size, agitation speed, working volume and induction profiling revealed that the fermentation conditions for the highest production of α-IFN2b (85.5 μg/L) was attained at inoculum size of 8%, working volume of 40% and agitation speed of 1000 rpm with induction at 4 h after the inoculation.

    CONCLUSION: Although the findings at MTP scale did not show perfect scalable results as compared to shake flask culture, but microscale technique development would serve as a convenient and low-cost solution in process optimization for recombinant protein.

    Matched MeSH terms: Recombinant Proteins/biosynthesis
  8. Fulltext Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris
    Gandhi S, Salleh AB, Rahman RN, Chor Leow T, Oslan SN
    Biomed Res Int, 2015;2015:529059.
    PMID: 26090417 DOI: 10.1155/2015/529059
    Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL(-1) at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg(-1). The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0-8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t₁/₂) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  9. Fulltext Optimization of a Bacillus sp signal peptide for improved recombinant protein secretion and cell viability in Escherichia coli: Is there an optimal signal peptide design?
    Low KO, Jonet MA, Ismail NF, Illias RM
    Bioengineered, 2012 Nov-Dec;3(6):334-8.
    PMID: 22892592 DOI: 10.4161/bioe.21454
    Recombinant protein fused to an N-terminal signal peptide can be translocated to the periplasm and, eventually, to the extracellular medium of Escherichia coli under specific conditions. In this communication, we described the use and optimization of a heterologous signal peptide (G1 signal peptide) from a Bacillus sp for improved recombinant protein secretion and cell viability in E. coli. Significant advantages in maintaining high cell viability and high specificity of target protein secretion were achieved by using G1 signal peptide compared to the well-known PelB signal peptide. Signal peptide sequence analysis and site-directed mutagenesis of G1 signal peptide demonstrated that an 'MKK' sequence in n-region and the presence of a helix-breaking residue at the centre of h-region are important elements for the design of an optimal signal peptide.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  10. Phage display antibodies for diagnostic applications
    Hairul Bahara NH, Tye GJ, Choong YS, Ong EB, Ismail A, Lim TS
    Biologicals, 2013 Jul;41(4):209-16.
    PMID: 23647952 DOI: 10.1016/j.biologicals.2013.04.001
    With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications. The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency. The flexibility to modify recombinant antibodies allows great applicability to various platforms for use. This review presents phage display technology, application and modifications of recombinant antibodies for diagnostics.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  11. Simplified feeding strategies for the fed-batch cultivation of Kluyveromyces lactis GG799 for enhanced recombinant xylanase production
    Fuzi SF, Razali F, Jahim JM, Rahman RA, Illias RM
    Bioprocess Biosyst Eng, 2014 Sep;37(9):1887-98.
    PMID: 24633311 DOI: 10.1007/s00449-014-1163-z
    A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20 g L(-1) and 80 % dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53 U mL(-1) was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13 U mL(-1)) was measured at a C/N ratio of 50.08. These conditions led to a 45.8 % increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500 g L(-1) glucose led to a 6.2-fold increase (465.07 U mL(-1)) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  12. Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment
    Abd Wahid MA, Megat Mohd Noor MJ, Goto M, Sugiura N, Othman N, Zakaria Z, et al.
    Biosci Biotechnol Biochem, 2017 Aug;81(8):1642-1649.
    PMID: 28585494 DOI: 10.1080/09168451.2017.1329617
    The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  13. Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris
    Woon JS, Mackeen MM, Mahadi NM, Illias RM, Abdul Murad AM, Abu Bakar FD
    Biotechnol Appl Biochem, 2016 Sep;63(5):690-698.
    PMID: 26265428 DOI: 10.1002/bab.1431
    The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic(®) CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  14. Human neonatal Fc receptor as a new potential antibody binding protein for antibody immobilization
    Ng WK, Lim TS, Lai NS
    Biotechnol Appl Biochem, 2018 Jul;65(4):547-553.
    PMID: 29280199 DOI: 10.1002/bab.1636
    A critical challenge in producing an antibody-based assay with the highest reproducibility and sensitivity is the strategy to immobilize antibodies to solid phase. To date, numerous methods of antibody immobilization were reported but each was subjected to its advantages and limitations. The current study proposes a new potential antibody binding protein, the human neonatal fragment crystallizable (Fc) receptor. This protein has shown its high affinity to the Fc of antibody either in vivo or in vitro. Human neonatal Fc receptor is a heterodimer constructed by p51 α-heavy chain and β2-microglobulin light chain; however, the binding sites toward the antibody are located in the p51 α-heavy chain. Hence, vector cloning and recombinant protein expression were carried out to express the p51 α-heavy chain of the human neonatal Fc receptor (hFcRn-α). The recombinant protein expressed, hFcRn-α, was adopted to pin rabbit IgG against hepatitis B virus surface antigen to a solid phase. A sandwich enzyme-linked immunosorbent assay was further developed to evaluate the efficiency of hFcRn-α-directed immobilization in antigen detection. The result was compared with the conventional physical adsorption method. The findings demonstrated that human neonatal Fc receptor was efficient in pinning antibodies and generating higher signals compared with the physical adsorption of antibody.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  15. 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/biosynthesis
  16. Strategies for humanizing glycosylation pathways and producing recombinant glycoproteins in microbial expression systems
    Khan AH, Noordin R
    Biotechnol Prog, 2019 03;35(2):e2752.
    PMID: 30457225 DOI: 10.1002/btpr.2752
    Homogeneously glycosylated proteins are essential for analyzing the structure of N-glycans, studying their consequences inside cells, and developing therapeutic glycoproteins. However, the isolation of glycoproteins with homogeneous glycans from human is difficult since glycoforms slightly differ from each other with respect to molecular weight and charge. Microbial expression systems have numerous benefits in expression technology and have gained great attention, because they are more adaptable to the biotechnology industry. While selecting an expression host, the glycosylation pattern must be taken into account, because glycosylation strongly depends on cellular production system and selected production clone. This review discussed the technological developments in glycoengineering of microbial expression systems for humanizing the glycosylation profile and highlighted the expression potential of Leishmania expression system. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2752, 2019.
    Matched MeSH terms: Recombinant Proteins/biosynthesis*
  17. Dengue envelope domain III-peptide binding analysis via tryptophan fluorescence quenching assay
    Baharuddin A, Amir Hassan A, Othman R, Xu Y, Huang M, Ario Tejo B, et al.
    Chem Pharm Bull (Tokyo), 2014;62(10):947-55.
    PMID: 25273053
    In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  18. Rational discovery of dengue type 2 non-competitive inhibitors
    Heh CH, Othman R, Buckle MJ, Sharifuddin Y, Yusof R, Rahman NA
    Chem Biol Drug Des, 2013 Jul;82(1):1-11.
    PMID: 23421589 DOI: 10.1111/cbdd.12122
    Various works have been carried out in developing therapeutics against dengue. However, to date, no effective vaccine or anti-dengue agent has yet been discovered. The development of protease inhibitors is considered as a promising option, but most previous works have involved competitive inhibition. In this study, we focused on rational discovery of potential anti-dengue agents based on non-competitive inhibition of DEN-2 NS2B/NS3 protease. A homology model of the DEN-2 NS2B/NS3 protease (using West Nile Virus NS2B/NS3 protease complex, 2FP7, as the template) was used as the target, and pinostrobin, a flavanone, was used as the standard ligand. Virtual screening was performed involving a total of 13 341 small compounds, with the backbone structures of chalcone, flavanone, and flavone, available in the ZINC database. Ranking of the resulting compounds yielded compounds with higher binding affinities compared with the standard ligand. Inhibition assay of the selected top-ranking compounds against DEN-2 NS2B/NS3 proteolytic activity resulted in significantly better inhibition compared with the standard and correlated well with in silico results. In conclusion, via this rational discovery technique, better inhibitors were identified. This method can be used in further work to discover lead compounds for anti-dengue agents.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  19. Potential application of Leishmania tarentolae as an alternative platform for antibody expression
    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: Recombinant Proteins/biosynthesis*
  20. A Critical Review of the Concept of Transgenic Plants: Insights into Pharmaceutical Biotechnology and Molecular Farming
    Abiri R, Valdiani A, Maziah M, Shaharuddin NA, Sahebi M, Yusof ZN, et al.
    Curr Issues Mol Biol, 2016;18:21-42.
    PMID: 25944541
    Using transgenic plants for the production of high-value recombinant proteins for industrial and clinical applications has become a promising alternative to using conventional bioproduction systems, such as bacteria, yeast, and cultured insect and animal cells. This novel system offers several advantages over conventional systems in terms of safety, scale, cost-effectiveness, and the ease of distribution and storage. Currently, plant systems are being utilised as recombinant bio-factories for the expression of various proteins, including potential vaccines and pharmaceuticals, through employing several adaptations of recombinant processes and utilizing the most suitable tools and strategies. The level of protein expression is a critical factor in plant molecular farming, and this level fluctuates according to the plant species and the organs involved. The production of recombinant native and engineered proteins is a complicated procedure that requires an inter- and multi-disciplinary effort involving a wide variety of scientific and technological disciplines, ranging from basic biotechnology, biochemistry, and cell biology to advanced production systems. This review considers important plant resources, affecting factors, and the recombinant-protein expression techniques relevant to the plant molecular farming process.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
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