Fatty acid desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated fatty acyl-chains to produce unsaturated fatty acids. A gene coding for a putative Δ9-fatty acid desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-fatty acid desaturase-like protein activity and increased overall levels of cellular unsaturated fatty acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic acid to oleic acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-fatty acid desaturase protein actively expressed in E. coli under the influence of temperature and an inducer.
Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.
DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, β-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform-isoamyl alcohol (24:1) or 6 M ammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).
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.
Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.
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.
Ornithine decarboxylase (ODC) is an enzyme of one of the two pathways of putrescine biosynthesis in plants. The genes encoding ODC have previously been cloned from Datura stramonium and human. Using differential screening, we isolated ODC cDNA clone from a cDNA library of ripening Capsicum annuum fruit. The cDNA clone designated CUKM10 contains an insert of 1523 bp. The longest open reading frame potentially encodes a peptide of 345 amino acids with an estimated molecular mass of 47 kDa and exhibit striking similarity to other ODCs. Expression analysis showed that the capODC hybridised to a single transcript with a size of 1.7 kb. The capODC transcript was first observed in early ripening and increased steadily until it reached fully ripening stage. From the observation it is suggested that capODC is developmentally regulated especially during later stage of ripening.
BACKGROUND:
Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information.
OBJECTIVE:
We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding.
METHODS:
The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients.
RESULTS:
The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue.
CONCLUSION:
The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.
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.
The front-end desaturases (Fads) are rate-limiting enzymes responsible for production of long-chain polyunsaturated fatty acids (LC-PUFA). The full spectrum of the transcriptional regulation of fads is still incomplete, as cloning of fads promoter is limited to a few species. Here, we described the cloning and characterisation of the zebrafish fads2 promoter. Using 5'-deletion and mutation analysis on this promoter, we identified a specific region containing the sterol regulatory element (SRE) which is responsible for the activation of the fads2 promoter. In tandem, two conserved CCAAT boxes were also present adjacent to the SRE and mutation of either of these binding sites attenuates the transcriptional activation of the fads2 promoter. An in vivo analysis employing GFP reporter gene in transiently transfected zebrafish embryos showed that this 1754 bp upstream region of the fads2 gene specifically directs GFP expression in the yolk syncytial layer (YSL) region. This indicates a role for LC-PUFA in the transport of yolk lipids through this tissue layer. In conclusion, besides identifying novel core elements for transcriptional activation in zebrafish fads2 promoter, we also reveal a potential role for fads2 or LC-PUFA in YSL during development.
In gram-negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N-acyl homoserine lactones (AHL). We have previously reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild-type E. asburiae. The mass spectrometry analysis showed the production of N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm-forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.
The Aspergillus flavus NSH9 gene, encoding a pH and thermostable glucoamylase with a starch binding domain (SBD), was expressed in Pichia pastoris to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp), and cDNA (1839 bp) encode a 612 amino acid protein most similar to glucoamylase from Aspergillus oryzae RIB40; the first 19 amino acids are presumed to be a signal peptide for secretion, and the SBD is at the C-terminal. The cDNA was successfully secreted by Pichia at 8.23 U mL-1, and the rGA2 was found to be: a 80 kDa monomer, stable from pH 3.0-9.0, with optimum catalytic activity at pH 5.0, active at temperatures up to 80°C (rGA2 retained 58% of its activity after 60 min of incubation at 70°C), and metal ions such as Na+, K+, Ca++ and Mg++ enhanced rGA2 enzyme activity. The starch degrading ability of rGA2 was also observed on raw sago starch and where prolonged incubation generated larger, deeper, holes on the starch granules, indicating rGA2 is an excellent candidate for industrial starch processing applications.
Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.
This study was established to test the hypothesis of whether the codon optimization of fish growth hormone gene (FGH) based on P. pastoris preferred codon will improve the quantity of secreted rFGH in culture supernatant that can directly be used as fish feed supplements. The optimized FGH coding sequence (oFGH) and native sequence (nFGH) of giant grouper fish (Epinephelus lanceolatus) were cloned into P. pastoris expression vector (pPICZαA) downstream of alcohol oxidase gene (AOX1) for efficient induction of extracellular rFGH by adding 1% of absolute methanol. The results showed that recombinant P. pastoris was able to produce 2.80 ± 0.27 mg of oFGH compared to 1.75 ± 0.25 of nFGH in one litre of culture supernatant. The total body weight of tiger grouper fingerlings fed with oFGH increased significantly at third (P < 0.05) and fourth weeks (P < 0.01) of four-week experiment period compared to those fed with nFGH. Both oFGH and nFGH significantly enhanced the final biomass and fish survival percentage. In conclusion, codon optimization of FGH fragment was useful to increase rFGH quantity in the culture supernatant of P. pastoris that can be directly used as fish feed supplements. Further studies are still required for large scale production of rFGH and practical application in aquaculture production.
Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.
Phytocystatin, a type of protease inhibitor (PI), plays major roles in plant defense mechanisms and has been reported to show antipathogenic properties and plant stress tolerance. Recombinant plant PIs are gaining popularity as potential candidates in engineering of crop protection and in synthesizing medicine. It is therefore crucial to identify PI from novel sources like Curcuma longa as it is more effective in combating against pathogens due to its novelty. In this study, a novel cDNA fragment encoding phytocystatin was isolated using degenerate PCR primers, designed from consensus regions of phytocystatin from other plant species. A full-length cDNA of the phytocystatin gene, designated CypCl, was acquired using 5'/3' rapid amplification of cDNA ends method and it has been deposited in NCBI database (accession number KF545954.1). It has a 687 bp long open reading frame (ORF) which encodes 228 amino acids. BLAST result indicated that CypCl is similar to cystatin protease inhibitor from Cucumis sativus with 74% max identity. Sequence analysis showed that CypCl contains most of the motifs found in a cystatin, including a G residue, LARFAV-, QxVxG sequence, PW dipeptide, and SNSL sequence at C-terminal extension. Phylogenetic studies also showed that CypCl is related to phytocystatin from Elaeis guineensis.
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.
The tachykinins are a family of neuropeptides, including substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), that are encoded by the tac1 (SP and NKA) or tac2/3 (NKB) genes. Tachykinins are widely distributed in the central nervous system and have roles as neurotransmitters and/or neuromodulators. Recent studies in mammals have demonstrated the coexpression of NKB and kisspeptin and their comodulatory roles over the control of reproduction. We have recently identified two kisspeptin-encoding genes, kiss1 and kiss2, in teleosts. However, such relationship between tachykinins and kisspeptins has not been demonstrated in non-mammalian species. To determine the involvement of tachykinins in the reproduction in teleosts, we identified tac1 and two tac2 (tac2a and tac2b) sequences in the zebrafish genome using in silico data mining. Zebrafish tac1 encodes SP and NKA, whereas the tac2 sequences encode NKB and an additional peptide homologous to NKB (NKB-related peptide). Digoxigenin in situ hybridization in the brain of zebrafish showed tac1 mRNA-containing cells in the olfactory bulb, telencephalon, preoptic region, hypothalamus, mesencephalon, and rhombencephalon. The zebrafish tac2a mRNA-containing cells were observed in the preoptic region, habenula, and hypothalamus, whereas the tac2b mRNA-containing cells were predominantly observed in the dorsal telencephalic area. Furthermore, we examined the coexpression of tachykinins and two kisspeptin genes in the brain of zebrafish. Dual fluorescent in situ hybridization showed no coexpression of tachykinins mRNA with kisspeptins mRNA in hypothalamic nuclei or the habenula. These results suggest the presence of independent pathways for kisspeptins and NKB neurons in the brain of zebrafish.
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%).