Displaying publications 1 - 20 of 219 in total

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  1. Rothan HA, Bahrani H, Shankar EM, Rahman NA, Yusof R
    Antiviral Res, 2014 Aug;108:173-80.
    PMID: 24929084 DOI: 10.1016/j.antiviral.2014.05.019
    Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
    Matched MeSH terms: Escherichia coli/genetics
  2. Rothan HA, Mohamed Z, Suhaeb AM, Rahman NA, Yusof R
    OMICS, 2013 Nov;17(11):560-7.
    PMID: 24044366 DOI: 10.1089/omi.2013.0056
    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.
    Matched MeSH terms: Escherichia coli/genetics
  3. Rothan HA, Bahrani H, Mohamed Z, Abd Rahman N, Yusof R
    PLoS One, 2014;9(4):e94561.
    PMID: 24722532 DOI: 10.1371/journal.pone.0094561
    Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.
    Matched MeSH terms: Escherichia coli/genetics
  4. Yap PS, Krishnan T, Chan KG, Lim SH
    J Microbiol Biotechnol, 2015 Aug;25(8):1299-306.
    PMID: 25381741 DOI: 10.4014/jmb.1407.07054
    This study aimed to investigate the mechanism of action of the cinnamon bark essential oil (CB), when used singly and also in combination with piperacillin, for its antimicrobial and synergistic activity against beta-lactamase TEM-1 plasmid-conferred Escherichia coli J53 R1. Viable count of this combination showed a complete killing profile at 20 h and further confirmed its synergistic effect by reducing the bacteria cell numbers. Analysis on the stability of treated cultures for cell membrane permeability by CB when tested against sodium dodecyl sulfate revealed that the bacterial cell membrane was disrupted by the essential oils. Scanning electron microscopy observation and bacterial surface charge measurement also revealed that CB causes irreversible membrane damage and reduces the bacterial surface charge. In addition, bioluminescence expression of Escherichia coli [pSB1075] and E. coli [pSB401] by CB showed reduction, indicating the possibility of the presence of quorum sensing (QS) inhibitors. Gas-chromatography and mass spectrometry of the essential oil of Cinnamomum verum showed that trans-cinnamaldehyde (72.81%), benzyl alcohol (12.5%), and eugenol (6.57%) were the major components in the essential oil. From this study, CB has the potential to reverse E. coli J53 R1 resistance to piperacillin through two pathways; modification in the permeability of the outer membrane or bacterial QS inhibition.
    Matched MeSH terms: Escherichia coli/genetics
  5. Kuah MK, Jaya-Ram A, Shu-Chien AC
    Biochim. Biophys. Acta, 2015 Mar;1851(3):248-60.
    PMID: 25542509 DOI: 10.1016/j.bbalip.2014.12.012
    The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.
    Matched MeSH terms: Escherichia coli/genetics
  6. Ranjani V, Janeček S, Chai KP, Shahir S, Abdul Rahman RN, Chan KG, et al.
    Sci Rep, 2014 Jul 28;4:5850.
    PMID: 25069018 DOI: 10.1038/srep05850
    The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, k(cat) and k(cat)/K(m) higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.
    Matched MeSH terms: Escherichia coli/genetics
  7. Goulter RM, Taran E, Gentle IR, Gobius KS, Dykes GA
    Colloids Surf B Biointerfaces, 2014 Jul 1;119:90-8.
    PMID: 24880987 DOI: 10.1016/j.colsurfb.2014.04.003
    The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.
    Matched MeSH terms: Adhesins, Escherichia coli/genetics
  8. Amerizadeh A, Khoo BY, Teh AY, Golkar M, Abdul Karim IZ, Osman S, et al.
    BMC Infect Dis, 2013;13:287.
    PMID: 23800344 DOI: 10.1186/1471-2334-13-287
    Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
    Matched MeSH terms: Escherichia coli/genetics
  9. Yuen CW, Ong EB, Mohamad S, Manaf UA, Najimudin N
    J Microbiol Biotechnol, 2012 Oct;22(10):1336-42.
    PMID: 23075783
    In Burkholderia pseudomallei, the pathogen that causes melioidosis, the gene cluster encoding the capsular polysaccharide, is located on chromosome 1. Among the 19 capsular genes in this cluster, wzm has not been thoroughly studied. To study the function of wzm, we generated a deletion mutant and compared it with the wild-type strain. The mutant produced less biofilm in minimal media and was more sensitive to desiccation and oxidative stress compared with the wild-type strain, indicating that wzm is involved in biofilm formation and membrane integrity. Scanning electron microscopy showed that the bacterial cells of the mutant strain have more defined surfaces with indentations, whereas cells of the wild-type strain do not.
    Matched MeSH terms: Escherichia coli/genetics
  10. Ho WS, Balan G, Puthucheary S, Kong BH, Lim KT, Tan LK, et al.
    Microb Drug Resist, 2012 Aug;18(4):408-16.
    PMID: 22394084 DOI: 10.1089/mdr.2011.0222
    The emergence of Escherichia coli resistant to extended-spectrum cephalosporins (ESCs) is of concern as ESC is often used to treat infections by Gram-negative bacteria. One-hundred and ten E. coli strains isolated in 2009-2010 from children warded in a Malaysian tertiary hospital were analyzed for their antibiograms, carriage of extended-spectrum beta-lactamase (ESBL) and AmpC genes, possible inclusion of the beta-lactamase genes on an integron platform, and their genetic relatedness. All E. coli strains were sensitive to carbapenems. About 46% of strains were multidrug resistant (MDR; i.e., resistant to ≥3 antibiotic classes) and almost half (45%) were nonsusceptible to ESCs. Among the MDR strains, high resistance rates were observed for ampicillin (98%), tetracycline (75%), and trimethoprim/sulfamethoxazole (73%). Out of 110 strains, bla(TEM-1) (49.1%), bla(CTX-M) (11.8%), and bla(CMY-2) (6.4%) were detected. Twenty-one strains were ESBL producers. CTX-M-15 was the predominant CTX-M variant found and this is the first report of a CTX-M-27-producing E. coli strain from Malaysia. Majority (3.1%) of the strains harbored class 1 integron-encoded integrases with a predominance of aadA and dfr genes within the integron variable region. No gene cassette encoding ESBL genes was found and integrons were not significantly associated with ESBL or non-ESBL producers. Possible clonal expansion was observed for few CTX-M-15-positive strains but the O25-ST131 E. coli clone known to harbor CTX-M-15 was not detected while CMY-2-positive strains were genetically diverse.
    Matched MeSH terms: Escherichia coli/genetics*
  11. Rahman RN, Salleh AB, Basri M, Wong CF
    Int J Mol Sci, 2011;12(9):5797-814.
    PMID: 22016627 DOI: 10.3390/ijms12095797
    Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
    Matched MeSH terms: Escherichia coli/genetics
  12. Levitskiy SA, Sycheva AM, Kharlampieva DD, Oberto J, Kamashev DE, Serebryakova MV, et al.
    Biochimie, 2011 Jul;93(7):1102-9.
    PMID: 21443922 DOI: 10.1016/j.biochi.2011.03.005
    HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.
    Matched MeSH terms: Escherichia coli/genetics
  13. Arockiaraj J, Easwvaran S, Vanaraja P, Singh A, Othman RY, Bhassu S
    Fish Shellfish Immunol, 2012 May;32(5):670-82.
    PMID: 22293093 DOI: 10.1016/j.fsi.2012.01.013
    In this study, we reported a full length of catalase gene (designated as MrCat), identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrCat is 2504 base pairs in length, and encodes 516 amino acids. The MrCat protein contains three domains such as catalase 1 (catalase proximal heme-ligand signature) at 350-358, catalase 2 (catalase proximal active site signature) at 60-76 and catalase 3 (catalase family profile) at 20-499. The mRNA expressions of MrCat in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). The MrCat is highly expressed in digestive tract and all the other tissues (walking leg, gills, muscle, hemocyte, hepatopancreas, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated in digestive tract after IHHNV challenge. To understand its biological activity, the recombinant MrCat gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCat existed in high thermal stability and broad spectrum of pH, which showed over 95% enzyme activity between pH 5 and 10.5, and was stable from 40 °C to 70 °C, and exhibited 85-100% enzyme activity from 30 °C to 40 °C.
    Matched MeSH terms: Escherichia coli/genetics
  14. Liew CW, Illias RM, Mahadi NM, Najimudin N
    FEMS Microbiol Lett, 2007 Nov;276(1):114-22.
    PMID: 17937670
    A Na(+)/H(+) antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na(+)/H(+) antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCl, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na(+) extrusion at lower pH values and in pH homeostasis at pH 10 under Na(+)-limiting conditions.
    Matched MeSH terms: Escherichia coli/genetics*
  15. Palasubramaniam S, Subramaniam G, Muniandy S, Parasakthi N
    Microb Drug Resist, 2007;13(3):186-90.
    PMID: 17949305
    In this report, we describe the detection of AmpC and CMY-2 beta-lactamases with the loss of OmpK35 porin among seven sporadic strains of ceftazidime-resistant Klebsiella pneumoniae and ceftazidime-resistant Escherichia coli. Cefoxitin, which was used as a marker of resistance toward 7-alpha-methoxy-cephalosporins, exhibited high minimum inhibitory concentration (MIC) values ranging between 128 microg/ml and >256 microg/ml in all the strains. The presence of hyperproducing AmpC enzymes was indicated by the positive three-dimensional test. Isoelectric focusing (IEF) study confirmed the presence of AmpC enzymes in all the strains. The ampC gene was detected by PCR in all the strains and confirmed by DNA sequencing. Large plasmids in all the strains, ranging from 60 kb to 150 kb in size, most likely encode the ampC gene. Two E. coli strains out of the seven strains showed positive amplification of the bla(CMY-2) gene, an AmpC variant, and was confirmed by DNA sequence analyses. DNA hybridization confirmed the bla(CMY-2) gene to be plasmid-mediated in both of these strains. However, one of these two strains also mediated a chromosomal CMY gene. All the strains showed an absence of OmpK35 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and was confirmed by western blot analyses using raised polyclonal anti-OmpK35 antiserum. This suggests that, apart from CMY production, absence of OmpK35 porin also contributed to cefoxitin resistance resulting in extended-spectrum beta-lactam resistance among these isolates.
    Matched MeSH terms: Escherichia coli/genetics
  16. Omar AR, Kim CL, Bejo MH, Ideris A
    J Vet Sci, 2006 Sep;7(3):241-7.
    PMID: 16871018
    The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.
    Matched MeSH terms: Escherichia coli/genetics
  17. Lee SS, Enchang FK, Tan NH, Fung SY, Pailoor J
    J Ethnopharmacol, 2013 May 2;147(1):157-63.
    PMID: 23458920 DOI: 10.1016/j.jep.2013.02.027
    Lignosus rhinocerus (Tiger Milk mushroom) is distributed in South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea. In Malaysia, it is the most popular medicinal mushroom used by the indigenous communities to relieve fever, cough, asthma, cancer, food poisoning and as a general tonic. In China, this mushroom is an expensive traditional medicine used to treat liver cancer, chronic hepatitis and gastric ulcers. The sclerotium of the mushroom is the part with medicinal value. This rare mushroom has recently been successfully cultivated making it possible to be fully exploited for its medicinal and functional benefits. The present study was carried out to evaluate the chronic toxicity of the sclerotial powder of Lignosus rhinocerus cultivar (termed TM02), its anti-fertility and teratogenic effects as well as genotoxicity.
    Matched MeSH terms: Escherichia coli/genetics
  18. Rothan HA, Ambikabothy J, Abdulrahman AY, Bahrani H, Golpich M, Amini E, et al.
    PLoS One, 2015;10(9):e0139248.
    PMID: 26418816 DOI: 10.1371/journal.pone.0139248
    The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.
    Matched MeSH terms: Escherichia coli/genetics
  19. Rabu A, Tan WS, Kho CL, Omar AR, Yusoff K
    Acta Virol., 2002;46(4):211-7.
    PMID: 12693857
    The nucleocapsid (NP) protein of Newcastle disease virus (NDV) self-assembled in Escherichia coli as ring-like and herringbone-like particles. Several chimeric NP proteins were constructed in which the antigenic regions of the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV, myc epitope, and six histidines (a hexa-His tag) were linked to the C-terminus of the NP monomer. These chimeric proteins were expressed efficiently in soluble form in E. coli as detected by Western blot analysis. Electron microscopy of the purified products revealed that they self-assembled into ring-like particles. These chimeric particles exhibited antigenicity of the myc epitope, suggesting that the foreign sequences were exposed on the surface of the particles. Chickens inoculated with the chimeric particles mounted an immune response against NDV, suggesting the possibility of use of the ring-like particle as a carrier of immunogens in subunit vaccines and immunological reagents.
    Matched MeSH terms: Escherichia coli/genetics
  20. 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: Escherichia coli/genetics*
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