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Fulltext Experimental Infection of Syrian Hamsters With Aerosolized Nipah Virus

Escaffre O, Hill T, Ikegami T, Juelich TL, Smith JK, Zhang L, et al.

J Infect Dis, 2018 10 05;218(10):1602-1610.
PMID: 29912426 DOI: 10.1093/infdis/jiy357

Background: Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited.
Methods: Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP).
Results: Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection.
Conclusion: Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.

Matched MeSH terms: Recombinant Proteins/metabolism
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/metabolism
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*
Facile modulation of enantioselectivity of thermophilic Geobacillus zalihae lipase by regulating hydrophobicity of its Q114 oxyanion

Abdul Wahab R, Basri M, Raja Abdul Rahman RN, Salleh AB, Abdul Rahman MB, Leow TC

Enzyme Microb Technol, 2016 Nov;93-94:174-181.
PMID: 27702478 DOI: 10.1016/j.enzmictec.2016.08.020

Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50°C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.

Matched MeSH terms: Recombinant Proteins/metabolism
Factors affecting therapeutic protein purity and yield during chromatographic purification

Che Hussian CHA, Leong WY

Prep Biochem Biotechnol, 2024 Feb;54(2):150-158.
PMID: 37233514 DOI: 10.1080/10826068.2023.2217507

Therapeutic proteins are recombinant proteins generated through recombinant DNA technology and have attracted a great deal of interest in numerous applications, including pharmaceutical, cosmetic, human and animal health, agriculture, food, and bioremediation. Producing therapeutic proteins on a large scale, mainly in the pharmaceutical industry, necessitates a cost-effective, straightforward, and adequate manufacturing process. In industry, a protein separation technique based mainly on protein characteristics and modes of chromatography will be applied to optimize the purification process. Typically, the downstream process of biopharmaceutical operations may involve multiple chromatography phases that require the use of large columns pre-packed with resins that must be inspected before use. Approximately 20% of the proteins are assumed to be lost at each purification stage during the production of biotherapeutic products. Hence, to produce a high quality product, particularly in the pharmaceutical industry, the correct approach and understanding of the factors influencing purity and yield during purification are necessary.

Matched MeSH terms: Recombinant Proteins/metabolism
Formation of new polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate monomer in Burkholderia sp

Lau NS, Tsuge T, Sudesh K

Appl Microbiol Biotechnol, 2011 Mar;89(5):1599-609.
PMID: 21279348 DOI: 10.1007/s00253-011-3097-6

Burkholderia sp. synthase has been shown to polymerize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate, and 3-hydroxy-4-pentenoic acid monomers. This study was carried out to evaluate the ability of Burkholderia sp. USM (JCM 15050) and its transformant harboring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae to incorporate the newly reported 3-hydroxy-4-methylvalerate (3H4MV) monomer. Various culture parameters such as concentrations of nutrient rich medium, fructose and 4-methylvaleric acid as well as harvesting time were manipulated to produce P(3HB-co-3H4MV) with different 3H4MV compositions. The structural properties of PHA containing 3H4MV monomer were investigated by using nuclear magnetic resonance and Fourier transform infrared spectroscopy (FTIR). The relative intensities of the bands at 1,183 and 1,228 cm⁻¹ in the FTIR spectra enabled the rapid detection and differentiation of P(3HB-co-3H4MV) from other types of PHA. In addition, the presence of 3H4MV units in the copolymer was found to considerably lower the melting temperature and enthalpy of fusion values compared with poly(3-hydroxybutyrate) (P(3HB)). The copolymer exhibited higher thermo-degradation temperature but similar molecular weight and polydispersity compared with P(3HB).

Matched MeSH terms: Recombinant Proteins/metabolism
Functional characterisation and product specificity of Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1

Jaafar NR, Khoiri NM, Ismail NF, Mahmood NAN, Abdul Murad AM, Abu Bakar FD, et al.

Enzyme Microb Technol, 2020 Oct;140:109625.
PMID: 32912685 DOI: 10.1016/j.enzmictec.2020.109625

Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.

Matched MeSH terms: Recombinant Proteins/metabolism
Gene expression and functional studies of small heat shock protein 37 (MrHSP37) from Macrobrachium rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus (IHHNV)

Arockiaraj J, Vanaraja P, Easwvaran S, Singh A, Othman RY, Bhassu S

Mol Biol Rep, 2012 Jun;39(6):6671-82.
PMID: 22290288 DOI: 10.1007/s11033-012-1473-7

In this study, we have reported a full length of small heat shock protein 37 (designated MrHSP37) gene, identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrHSP37 is 2,425 base pairs in length, and encodes 338 amino acids. MrHSP37 contains a long heat shock protein family profile in the amino acid sequence between 205 and 288. The mRNA expressions of MrHSP37 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). MrHSP37 is highly expressed in hepatopancreas and all the other tissues (walking leg, gills, muscle, stomach, haemocyte, intestine, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated after IHHNV challenge. To understand its biological activity, the recombinant MrHSP37 gene was constructed and expressed in Escherichia coli BL21 (DE3). The results of ATPase assay showed that the recombinant MrHSP37 protein exhibited apparent ATPase activity which increased with the concentration of the protein. And also the purified recombinant MrHSP37 protein was used for thermal aggregation assay (chaperone activity). It showed that the recombinant MrHSP37 protein is an active chaperone in this assay. Taken together, these results suggest that MrHSP37 is potentially involved in the immune responses against IHHNV challenge in M. rosenbergii.

Matched MeSH terms: Recombinant Proteins/metabolism
Fulltext Heterologous Expression of a Novel Drug Transporter from the Malaria Parasite Alters Resistance to Quinoline Antimalarials

Tindall SM, Vallières C, Lakhani DH, Islahudin F, Ting KN, Avery SV

Sci Rep, 2018 02 06;8(1):2464.
PMID: 29410428 DOI: 10.1038/s41598-018-20816-0

Antimalarial drug resistance hampers effective malaria treatment. Critical SNPs in a particular, putative amino acid transporter were recently linked to chloroquine (CQ) resistance in malaria parasites. Here, we show that this conserved protein (PF3D7_0629500 in Plasmodium falciparum; AAT1 in P. chabaudi) is a structural homologue of the yeast amino acid transporter Tat2p, which is known to mediate quinine uptake and toxicity. Heterologous expression of PF3D7_0629500 in yeast produced CQ hypersensitivity, coincident with increased CQ uptake. PF3D7_0629500-expressing cultures were also sensitized to related antimalarials; amodiaquine, mefloquine and particularly quinine. Drug sensitivity was reversed by introducing a SNP linked to CQ resistance in the parasite. Like Tat2p, PF3D7_0629500-dependent quinine hypersensitivity was suppressible with tryptophan, consistent with a common transport mechanism. A four-fold increase in quinine uptake by PF3D7_0629500 expressing cells was abolished by the resistance SNP. The parasite protein localised primarily to the yeast plasma membrane. Its expression varied between cells and this heterogeneity was used to show that high-expressing cell subpopulations were the most drug sensitive. The results reveal that the PF3D7_0629500 protein can determine the level of sensitivity to several major quinine-related antimalarials through an amino acid-inhibitable drug transport function. The potential clinical relevance is discussed.

Matched MeSH terms: Recombinant Proteins/metabolism
Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker

Pan Y, Mak JW, Ong CE

Biomed Chromatogr, 2013 Jul;27(7):859-65.
PMID: 23386533 DOI: 10.1002/bmc.2872

In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP-HPLC assay showed good linearity (r(2) = 1.00) with 4-hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10(-2) μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km , Vmax and Ki ) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.

Matched MeSH terms: Recombinant Proteins/metabolism*
Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization

Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Ong CE

Protein J, 2011 Dec;30(8):581-91.
PMID: 22001938 DOI: 10.1007/s10930-011-9365-6

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.

Matched MeSH terms: Recombinant Proteins/metabolism
Heterologous expression of the tetracycline resistance gene tetX to enhance degradability and safety in doxycycline degradation

Wen X, Huang J, Cao J, Xu J, Mi J, Wang Y, et al.

Ecotoxicol Environ Saf, 2020 Mar 15;191:110214.
PMID: 31968275 DOI: 10.1016/j.ecoenv.2020.110214

Microbial remediation has the potential to inexpensively yet effectively decontaminate and restore contaminated environments, but the virulence of pathogens and risk of resistance gene transmission by microorganisms during antibiotic removal often limit its implementation. Here, a cloned tetX gene with clear evolutionary history was expressed to explore doxycycline (DOX) degradation and resistance variation during the degradation process. Phylogenetic analysis of tetX genes showed high similarity with those of pathogenic bacteria, such as Riemerella sp. and Acinetobacter sp. Successful tetX expression was performed in Escherichia coli and confirmed by SDS-PAGE and Western blot. Our results showed that 95.0 ± 1.0% of the DOX (50 mg/L) was degraded by the recombinant strain (ETD-1 with tetX) within 48 h, which was significantly higher than that for the control (38.9 ± 8.7%) and the empty plasmid bacteria (8.8 ± 5.1%) (P 0.05). The efficient and safe DOX-degrading capacity of the recombinant strain ETD-1 makes it valuable and promising for antibiotic removal in the environment.

Matched MeSH terms: Recombinant Proteins/metabolism
Heterologous expression, purification and biochemical characterization of a new endo-1,4-β-xylanase from Rhodothermaceae bacterium RA

Liew KJ, Ngooi CY, Shamsir MS, Sani RK, Chong CS, Goh KM

Protein Expr Purif, 2019 12;164:105464.
PMID: 31376486 DOI: 10.1016/j.pep.2019.105464

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

Matched MeSH terms: Recombinant Proteins/metabolism
Fulltext Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris

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: Recombinant Proteins/metabolism
High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions

Seman WM, Bakar SA, Bukhari NA, Gaspar SM, Othman R, Nathan S, et al.

J Biotechnol, 2014 Aug 20;184:219-28.
PMID: 24910973 DOI: 10.1016/j.jbiotec.2014.05.034

A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.

Matched MeSH terms: Recombinant Proteins/metabolism
Humanizing glycosylation pathways in eukaryotic expression systems

Khan AH, Bayat H, Rajabibazl M, Sabri S, Rahimpour A

World J Microbiol Biotechnol, 2017 Jan;33(1):4.
PMID: 27837408

Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system.

Matched MeSH terms: Recombinant Proteins/metabolism
Identification and functional analysis of variants in the human concentrative nucleoside transporter 2, hCNT2 (SLC28A2) in Chinese, Malays and Indians

Li L, Tan CM, Koo SH, Chong KT, Lee EJ

Pharmacogenet Genomics, 2007 Sep;17(9):783-6.
PMID: 17700367

The human concentrative nucleoside transporter (hCNT2), also known as SLC28A2, plays an important role in the cellular uptake across intestinal membrane of some naturally occurring nucleosides and nucleoside analogs. This study aims to determine the genetic variability of hCNT2 (SLC28A2) in three major Asian ethnic groups residing in Singapore: Chinese, Malay and Indian, and functionally characterize the variants of hCNT2. Healthy participants (n=96) from each group were screened for genetic variations in the exons of hCNT2 (SLC28A2) using denaturing high performance liquid chromatography and sequencing analyses. A total of 23 polymorphisms were identified in the exonic and flanking intronic regions, and ethnic differences in single nucleotide polymorphism frequencies were evident. Five novel nonsynonymous variants (L12R, R142H, E172D, E385K, M612T) were constructed by mutagenesis and functionally characterized in U-251 cells. Expression of these variants in U-251 cells revealed that all except E385K can uptake various substrates of hCNT2: inosine, ribavirin and uridine.

Matched MeSH terms: Recombinant Proteins/metabolism
Fulltext Identification and real-time expression analysis of selected Toxoplasma gondii in-vivo induced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients

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: Recombinant Proteins/metabolism
Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1

Atago Y, Shimodaira J, Araki N, Bin Othman N, Zakaria Z, Fukuda M, et al.

Biosci Biotechnol Biochem, 2016 May;80(5):1012-9.
PMID: 26828632 DOI: 10.1080/09168451.2015.1127134

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

Matched MeSH terms: Recombinant Proteins/metabolism
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

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