The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min.
We have characterized an oil palm (Elaeis guineensis Jacq.) constitutive promoter that is derived from a translationally control tumor protein (TCTP) gene. The TCTP promoter was fused transcriptionally with the gusA reporter gene and transferred to monocot and dicot systems in order to study its regulatory role in a transient expression study. It was found that the 5' region of TCTP was capable of driving the gusA expression in all the oil palm tissues tested, including immature embryo, embryogenic callus, embryoid, young leaflet from mature palm, green leaf, mesocarp and stem. It could also be used in dicot systems as it was also capable of driving gusA expression in tobacco leaves. The results indicate that the TCTP promoter could be used for the production of recombinant proteins that require constitutive expression in the plant system.
Rhoptry protein 2 (ROP2) of Toxoplasma gondii is a rhoptry-secreted protein that plays a critical role in parasitophorous vacuole membrane formation during invasion. In previous studies, ROP2 has been shown to be efficient in triggering humoral and cell-mediated responses. High immunogenicity of ROP2 makes it a potential candidate for diagnosis and vaccination against toxoplasmosis. In this study, the ROP2 gene was cloned into pPICZα A expression vector and extracellularly expressed in the yeast Pichia pastoris, which has numerous advantages over other expression systems for eukaryotic proteins expression. The effectiveness of the secreted recombinant ROP2 as a diagnosis agent was assessed by Western Blot with 200 human serum samples. Recombinant ROP2 reacted with toxoplasmosis-positive human serum samples and yielded an overall sensitivity of 90% and specificity of 95%. However, recombinant ROP2 is a better marker for detection of IgG (91.7%) rather than IgM (80%).
Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.
Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.
Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C(6)-C(14)) to produce tri- and tetraketide pyrones. Mutations at H(331) and N(364) caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His(227) and Leu(366) play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.
An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.
Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.
Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.
Plastid trnL-trnF and nuclear ribosomal ITS sequences were obtained from selected wild-type individuals of Polygonum minus Huds. in Peninsular Malaysia. The 380 bp trnL-trnF sequences of the Polygonum minus accessions were identical. Therefore, the trnL-trnF failed to distinguish between the Polygonum minus accessions. However, the divergence of ITS sequences (650 bp) among the Polygonum minus accessions was 1%, indicating that these accessions could be distinguished by the ITS sequences. A phylogenetic relationship based on the ITS sequences was inferred using neighbor-joining, maximum parsimony and Bayesian inference. All of the tree topologies indicated that Polygonum minus from Peninsular Malaysia is unique and different from the synonymous Persicaria minor (Huds.) Opiz and Polygonum kawagoeanum Makino.
Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.
Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).
Detection of Toxoplasma gondii infection is essential in pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several Toxoplasma antigens, including dense granule antigens (GRAs) has high potential as diagnostic reagents. In the present study, we produced GRA2 using Pichia pastoris system. RNA of T. gondii RH strain tachyzoite was used as a template to produce cDNA clones of full-length GRA2 via reverse transcriptase PCR. Amplicons were inserted into pPICZalpha A and the recombinant plasmid transformed into P. pastoris, X-33 strain. The expressed recombinant protein was identified by SDS-PAGE and Western blotting. A recombinant protein of -28 kDa was produced, which could be detected by toxoplasmosis positive human sera indicating that the recombinant protein retained its antigenicity. The present study indicates that P. pastoris-expressed GRA2 should be useful for detection of Toxoplasma infection.
Galactose-1-phosphate uridylyltransferase (GALT) catalyzes the reversible conversion of glucose-1-phosphate and UDP-galactose to galactose-1-phosphate and UDP-glucose. This enzyme is also responsible for one of the biochemical steps that produce the precursors of agar and agarose. In this study, we report the molecular cloning and sequence analyses of a cDNA encoding GALT, from Gracilaria changii (B. M. Xia et I. A. Abbott) I. A. Abbott, J. Zhang et B. M. Xia, which constitutes a genus of seaweeds that supply more than 60% of the world's agar and agarose. We have subcloned this cDNA into a bacterial expression cloning vector and characterized the enzyme activities of its recombinant proteins in vitro. The GcGALT gene was shown to be up-regulated by salinity stresses. The abundance of transcripts encoding GcGALT was the highest in G. changii, followed by Gracilaria edulis and Gracilaria salicornia in a descending order, corresponding to their respective agar contents. Our findings indicated that GALT could be one of the components that determines the agar yield in Gracilaria species.
MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21-24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.
In addition to reproduction, gonadotropin-releasing hormone (GnRH) has been postulated to control cholesterol metabolism via cholesterol transport, which is carried out partly by the members of ATP-binding cassette (ABC) transporters G1 (ABCG1) and G4 (ABCG4). However, there is yet to be evidence demonstrating the relationship between these transporters with reference to GnRH neurons. In the present study, we cloned two ABCG1 messenger RNA (mRNA) variants and one ABCG4 mRNA and examined their expression in the brain including GnRH neurons (GnRH1, GnRH2, and GnRH3) in the cichlid tilapia (Oreochromis niloticus). Comparison of nucleotide sequences of the tilapia ABCG1 and ABCG4 with that of other fish species showed that both of these genes are evolutionarily conserved among fishes. ABCG1 and ABCG4 were shown to have high mRNA expressions in the CNS, pituitary, and gonads. In the brain, real-time polymerase chain reaction (PCR) showed that ABCG4 mRNA was higher than ABCG1a in all brain regions including the olfactory bulb (ABCG1=13.34, ABCG4=6796.35; P<0.001), dorsal telencephalon (ABCG1=8.64, ABCG4=10149.13; P=0.001), optic tectum (ABCG1=22.12, ABCG4=13931.04; P<0.01), cerebellum (ABCG1=8.68, ABCG4=12382.90; P<0.01), and preoptic area-midbrain-hypothalamus (ABCG1=21.36, ABCG4=13255.41; P=0.001). Similarly, although ABCG1 mRNA level is much higher in the pituitary compared with the brain, it was still significantly lower compared with ABCG4 (ABCG1=337.73, ABCG4=1157.87; P=0.01). The differential pattern of expression of ABCG1 and ABCG4 in the brain versus pituitary suggests that the two transporters are regulated by different mechanisms. Furthermore, ABCG1 and ABCG4 mRNA expressions were found in all three types of laser-captured GnRH neurons with highly similar percentage of expressions, suggesting that cholesterol efflux from GnRH neurons may require heterodimerization of both ABCG1 and ABCG4.
In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
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.
Alcanivorax hongdengensis A-11-3 is a newly identified type strain isolated from the surface water of the Malacca and Singapore Straits that can degrade a wide range of alkanes. To understand the degradation mechanism of this strain, the genes encoding alkane hydroxylases were obtained by PCR screening and shotgun sequencing of a genomic fosmid library. Six genes involved in alkane degradation were found, including alkB1, alkB2, p450-1, p450-2, p450-3 and almA. Heterogeneous expression analysis confirmed their functions as alkane oxidases in Pseudomonas putida GPo12 (pGEc47ΔB) or Pseudomonas fluorescens KOB2Δ1. Q-PCR revealed that the transcription of alkB1 and alkB2 was enhanced in the presence of n-alkanes C(12) to C(24); three p450 genes were up-regulated by C(8)-C(16) n-alkanes at different levels, whereas enhanced expression of almA was observed when strain A-11-3 grew with long-chain alkanes (C(24) to C(36)). In the case of branched alkanes, pristane significantly enhanced the expression of alkB1, p450-3 and almA. The six genes enable strain A-11-3 to degrade short (C(8)) to long (C(36)) alkanes that are straight or branched. The ability of A. hongdengensis A-11-3 to thrive in oil-polluted marine environments may be due to this strain's multiple systems for alkane degradation and its range of substrates.
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.