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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/isolation & purification; Recombinant Proteins/metabolism
Fulltext A comparative study on the expression, purification and functional characterization of human adiponectin in Pichia pastoris and Escherichia coli

Rothan HA, Teh SH, Haron K, Mohamed Z

Int J Mol Sci, 2012;13(3):3549-62.
PMID: 22489167 DOI: 10.3390/ijms13033549

Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.

Matched MeSH terms: Recombinant Proteins/biosynthesis*; Recombinant Proteins/genetics
Fulltext Functional expression of an orchid fragrance gene in Lactococcus lactis

Song AA, Abdullah JO, Abdullah MP, Shafee N, Rahim RA

Int J Mol Sci, 2012;13(2):1582-97.
PMID: 22408409 DOI: 10.3390/ijms13021582

Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile.

Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics
Fulltext Lactococcus lactis M4, a potential host for the expression of heterologous proteins

Noreen N, Hooi WY, Baradaran A, Rosfarizan M, Sieo CC, Rosli MI, et al.

Microb Cell Fact, 2011;10:28.
PMID: 21518457 DOI: 10.1186/1475-2859-10-28

Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.

Matched MeSH terms: Recombinant Proteins/biosynthesis*; Recombinant Proteins/genetics
A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli

Ismail NF, Hamdan S, Mahadi NM, Murad AM, Rabu A, Bakar FD, et al.

Biotechnol Lett, 2011 May;33(5):999-1005.
PMID: 21234789 DOI: 10.1007/s10529-011-0517-8

L-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS-PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/secretion
Characterization of secreted recombinant Toxoplasma gondii surface antigen 2 (SAG2) heterologously expressed by the yeast Pichia pastoris

Fong MY, Lau YL, Zulqarnain M

Biotechnol Lett, 2008 Apr;30(4):611-8.
PMID: 18043869

The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii.

Matched MeSH terms: Recombinant Proteins/immunology; Recombinant Proteins/metabolism*
Direct purification of recombinant hepatitis B core antigen from two different pre-conditioned unclarified Escherichia coli feedstocks via expanded bed adsorption chromatography

Ng MY, Tan WS, Abdullah N, Ling TC, Tey BT

J Chromatogr A, 2007 Nov 16;1172(1):47-56.
PMID: 17945242

Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.

Matched MeSH terms: Recombinant Proteins/isolation & purification*; Recombinant Proteins/chemistry
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/genetics; Recombinant Proteins/metabolism
Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene

Ishikawa T, Abe M, Masuda M

Virus Res, 2015 Jan 2;195:153-61.
PMID: 25451067 DOI: 10.1016/j.virusres.2014.10.010

Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V.

Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics
Fulltext New Recombinant Cold-Adapted and Organic Solvent Tolerant Lipase from Psychrophilic Pseudomonas sp. LSK25, Isolated from Signy Island Antarctica

Salwoom L, Raja Abd Rahman RNZ, Salleh AB, Mohd Shariff F, Convey P, Mohamad Ali MS

Int J Mol Sci, 2019 Mar 13;20(6).
PMID: 30871178 DOI: 10.3390/ijms20061264

In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism*
Fulltext Potential use of Pichia pastoris strain SMD1168H expressing DNA topoisomerase I in the screening of potential anti‑breast cancer agents

Xin J, Wan Mahtar WNA, Siah PC, Miswan N, Khoo BY

Mol Med Rep, 2019 Jun;19(6):5368-5376.
PMID: 31059050 DOI: 10.3892/mmr.2019.10201

Cancer chemotherapy possesses high toxicity, particularly when a higher concentration of drugs is administered to patients. Therefore, searching for more effective compounds to reduce the toxicity of treatments, while still producing similar effects as current chemotherapy regimens, is required. Currently, the search for potential anticancer agents involves a random, inaccurate process with strategic deficits and a lack of specific targets. For this reason, the initial in vitro high‑throughput steps in the screening process should be reviewed for rapid identification of the compounds that may serve as anticancer agents. The present study aimed to investigate the potential use of the Pichia pastoris strain SMD1168H expressing DNA topoisomerase I (SMD1168H‑TOPOI) in a yeast‑based assay for screening potential anticancer agents. The cell density that indicated the growth of the recombinant yeast without treatment was first measured by spectrophotometry. Subsequently, the effects of glutamate (agonist) and camptothecin (antagonist) on the recombinant yeast cell density were investigated using the same approach, and finally, the effect of camptothecin on various cell lines was determined and compared with its effect on recombinant yeast. The current study demonstrated that growth was enhanced in SMD1168H‑TOPOI as compared with that in SMD1168H. Glutamate also enhanced the growth of the SMD1168H; however, the growth effect was not enhanced in SMD1168H‑TOPOI treated with glutamate. By contrast, camptothecin caused only lower cell density and growth throughout the treatment of SMD1168H‑TOPOI. The findings of the current study indicated that SMD1168H‑TOPOI has similar characteristics to MDA‑MB‑231 cells; therefore, it can be used in a yeast‑based assay to screen for more effective compounds that may inhibit the growth of highly metastatic breast cancer cells.

Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics
Cost-utility analysis of an adjunctive recombinant activated factor VIIa for on-demand treatment of bleeding episodes in dengue haemorrhagic fever

Naing C, Poovorawan Y, Mak JW, Aung K, Kamolratankul P

Blood Coagul Fibrinolysis, 2015 Jun;26(4):403-7.
PMID: 25692521 DOI: 10.1097/MBC.0000000000000280

The present study aimed to assess the cost-utility analysis of using an adjunctive recombinant activated factor VIIa (rFVIIa) in children for controlling life-threatening bleeding in dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS). We constructed a decision-tree model, comparing a standard care and the use of an additional adjuvant rFVIIa for controlling life-threatening bleeding in children with DHF/DSS. Cost and utility benefit were estimated from the societal perspective. The outcome measure was cost per quality-adjusted life years (QALYs). Overall, treatment with adjuvant rFVIIa gained QALYs, but the total cost was higher. The incremental cost-utility ratio for the introduction of adjuvant rFVIIa was $4241.27 per additional QALY. Sensitivity analyses showed the utility value assigned for calculation of QALY was the most sensitive parameter. We concluded that despite high cost, there is a role for rFVIIa in the treatment of life-threatening bleeding in patients with DHF/DSS.

Matched MeSH terms: Recombinant Proteins/economics; Recombinant Proteins/therapeutic use
Fulltext Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Norsyahida A, Rahmah N, Ahmad RM

Lett Appl Microbiol, 2009 Nov;49(5):544-50.
PMID: 19832937 DOI: 10.1111/j.1472-765X.2009.02694.x

To investigate the effects of feeding and induction strategies on the production of BmR1 recombinant antigen.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism
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; Recombinant Proteins/genetics
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; Recombinant Proteins/genetics
Fulltext Recombinant proteins from new constructs of SAG1 and GRA7 sequences and their usefulness to detect acute toxoplasmosis

Kotresha D, Poonam D, Muhammad Hafiznur Y, Saadatnia G, Nurulhasanah O, Sabariah O, et al.

Trop Biomed, 2012 Mar;29(1):129-37.
PMID: 22543613 MyJurnal

In this study we have cloned unreported gene fragments of Toxoplasma gondii GRA7 and SAG1 and expressed the corresponding recombinant proteins, followed by evaluation of their usefulness for the serological diagnosis of toxoplasmosis. Both recombinant proteins were expressed efficiently in insoluble form, purified by single step Ni-NTA affinity chromatography and their antigenicity to detect toxoplasma specific IgG antibodies were determined by immunoblotting. A total of 60 serum samples from three groups of individuals based on their anti-toxoplasma antibody profiles were tested, namely (I) IgM+, IgG+ (n=20), (II) IgM-, IgG+ (n=20) and (III) IgM-, IgG- (n=20). Both recombinant proteins exhibited high sensitivity (100%) with sera from Group I. rGRA7 and rSAG1 reacted 40% and 80% respectively with Group II sera. The specificity of the recombinant proteins based on reactivities with Group III sera were 100% and 80% with rGRA7 and rSAG1 respectively. Thus rGRA7 was found to be better at discriminating probable acute from chronic phases of toxoplasmosis, and it also showed higher specificity.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification
Fulltext Phagocytic activity and pro-inflammatory cytokines production by the murine macrophage cell line J774A.1 stimulated by a recombinant BCG (rBCG) expressing the MSP1-C of Plasmodium falciparum

Rapeah S, Dhaniah M, Nurul AA, Norazmi MN

Trop Biomed, 2010 Dec;27(3):461-9.
PMID: 21399587 MyJurnal

Macrophages are involved in innate immunity against malaria due to their ability to phagocytose infected erythrocytes and produce inflammatory cytokines, which are important for controlling parasite growth during malaria infection. In this study, the ability of a recombinant BCG (rBCG) vaccine expressing the 19-kDa C-terminus of merozoite surface protein-1 (MSP1-C) of Plasmodium falciparum, to stimulate the phagocytic activity and secretion of pro-inflammatory cytokines by the macrophage cell line J774A.1 was measured at varying times. The results demonstrate the ability of the rBCG construct to activate the inflammatory action of macrophages, which is important as a first-line of defence in clearing malaria infections.

Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology
Pharmacokinetics of a novel extended half-life glycoPEGylated factor IX, nonacog beta pegol (N9-GP) in previously treated patients with haemophilia B: results from two phase 3 clinical trials

Tiede A, Abdul-Karim F, Carcao M, Persson P, Clausen WHO, Kearney S, et al.

Haemophilia, 2017 Jul;23(4):547-555.
PMID: 28233381 DOI: 10.1111/hae.13191

INTRODUCTION: Nonacog beta pegol (N9-GP) is a glycoPEGylated recombinant factor IX (FIX) with an extended half-life developed for routine prophylaxis and the prevention and treatment of bleeding episodes in patients with haemophilia B.
AIM: The aim of this study was to evaluate the pharmacokinetics (PK) of N9-GP.
METHODS: Data from 41 previously treated haemophilia B patients, enrolled globally (16 adolescents/adults and 25 children; FIX activity ≤0.02 IU mL-1) with no history of FIX inhibitors, were included. N9-GP was administered once-weekly as 10 IU kg-1or 40 IU kg-1in adolescents/adults and 40 IU kg-1in children. Blood was sampled up to 168 h (1 week) post dose. Standard PK was estimated on the basis of plasma FIX activity vs. time (PK profiles) using non-compartmental methods. Furthermore, a population PK analysis and FIX activity predictions were performed.
RESULTS: Incremental recoveries were 0.02 (IU mL-1)/(IU kg-1) in both adolescents/adults and children. The extended half-life resulted in mean trough levels of 0.27 IU mL-1for adolescents/adults and 0.17 IU mL-1for children at steady-state after weekly dosing at 40 IU kg-1. The population PK analysis confirmed a mono-exponential decay in FIX activity and allowed for predictions of FIX activity for adolescents/adults above 0.15 IU mL-1at all times and 6.4 days week-1in children.
CONCLUSION: N9-GP has the potential to shift previously treated haemophilia B patients from a severe/moderate disease state into a mild- or non-haemophilic range for most of the dosing interval, which is expected to reduce the number of bleeding episodes.

Matched MeSH terms: Recombinant Proteins/pharmacokinetics; Recombinant Proteins/therapeutic use
Fulltext Characterizing a Halo-Tolerant GH10 Xylanase from Roseithermus sacchariphilus Strain RA and Its CBM-Truncated Variant

Teo SC, Liew KJ, Shamsir MS, Chong CS, Bruce NC, Chan KG, et al.

Int J Mol Sci, 2019 May 09;20(9).
PMID: 31075847 DOI: 10.3390/ijms20092284

A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2ΔCBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2ΔCBM had an identical optimum temperature at 70 °C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2ΔCBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s-1, but this was reduced by 40% for XynRA2ΔCBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.

Matched MeSH terms: Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
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/genetics; Recombinant Proteins/metabolism

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