31 research outputs found
Expression and characterization of full length and truncated versions of major outercapsid protein VP2 of bluetongue virus in bacterial and insect cells
The spread of bluetongue virus (BTV) to previously disease-free regions which prohibit the use of the current BTV live-attenuated vaccine has highlighted the need for a new generation of vaccines (Ferrari, De Liberato et al. 2005; Veronesi, Hamblin et al. 2005). Subunit vaccines are one of the attractive alternative strategies. Subunit vaccines against BTV would target the outercapsid protein VP2, the main neutralization-specific antigen (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). A subunit vaccine based on the use of BTV-VP2 may be achieved by either using VP2 by itself or by means of virus-like particles (VLPs) on which VP2 proteins are exposed. In VLPs, the VP2 is co-expressed with other capsid and core proteins to form a particle that resembles the intact BTV. The BTV-VLP vaccine strategy is advantageous since it presents the neutralizing epitopes of more than one viral protein in a more authentic manner as found on the virus itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However there are difficulties associated with large scale production and a decrease in the stability of the particles over time (Berg, Difatta et al. 2005; Wang, Zhao et al. 2006). Studies have already demonstrated the vaccine potential of BTVVP2 by itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However if BTV-VP2 is to be used by itself as a single subunit vaccine, it is important that the protein is expressed under conditions where it is correctly folded and soluble. Solubility refers to the capacity of the expressed antigen to fold into an ordered tertiary structure that authentically exposes the neutralizing epitopes to the immune system (Dinner, Sali et al. 2000; Dobson 2003). However non-native interactions within and between in vitro synthesized viral proteins such as BTV-VP2 often leads to protein aggregation or insolubility. The immune response against aggregated or insoluble proteins is generally very poor. This problem of aggregation and insolubility may be alleviated to an extent by generating truncated versions of the protein from which hydrophobic regions that promote aggregation have been deleted leaving only the major neutralizing epitopes of the antigen (Fukumoto, Xuan et al. 2003; Bonafe, Rininger et al. 2009; Liu, Zeng et al. 2009; Seo, Pyo et al. 2009). The focus of the research presented in this dissertation was to evaluate the solubility of full-length BTV(10)-VP2 and truncated versions thereof after expression in a prokaryotic and baculovirus-Sf9 expression system. The full-length BTV(10)-VP2 (956 amino acids) gene and genes encoding truncated versions of BTV(10)-VP2 i.e. BTV(10)-VP2(aa450) (amino acid 1 to 450) and BTV(10)- VP2(aa650) (amino acid 1 to 650) were cloned into the bacterial expression vector pET160-DEST and the baculovirus expression vector pDEST™8. The C-terminal hydrophobic regions which might contribute to aggregation or insolubility of the protein when expressed in vitro were deleted from these truncated BTV(10)-VP2 proteins. The truncated proteins however still contained BTV neutralizing epitopes that were predicted from literature. The prokaryotic expression of the full-length BTV(10)-VP2 and the other truncated recombinant BTV(10)-VP2 proteins was carried out in E. coli BL21 Star DE3 expression strain. The initial pilot expression study confirmed high level expression of the recombinant proteins. The study also revealed that these proteins were insoluble. The optimization of the prokaryotic expression in order to increase the yield of soluble proteins by means of differential inducer concentrations, fermentation temperature and harvesting times did not produce soluble BTV(10)-VP2 and truncated BTV(10)-VP2 proteins. Previous studies have demonstrated the role of L-arginine in the recovery of soluble proteins from aggregation by reversing aggregation (Tsumoto, Umetsu et al. 2003). However in the current study, arginine treatment of the inclusion body and bacterial lysate containing the BTV(10)- VP2 and truncated recombinant proteins did not release soluble proteins. No soluble recombinant BTV(10)-VP2 proteins were detected when the recombinant proteins were expressed in BL21 host cells over-expressing heat-shock proteins (hsps) and chemical chaperones. However when the different recombinant proteins were co-expressed with the molecular chaperones dnaK-dnaJ-GrpE, it resulted in a fraction of soluble recombinant BTV(10)-VP2 proteins. In particular, approximately 50% of the total expressed BTV(10)-VP2(aa450) protein was soluble while approximately 20% of the total expressed BTV(10)-VP2(aa650) and full-length BTV(10)-VP2 were found soluble when coexpressed with dnaK-dnaJ-GrpE chaperones. These recombinant proteins could be eluted from a nickel affinity column further confirming that these proteins are in fact soluble. Interestingly the coexpression of the BTV(10)-VP2(aa450) protein with the above chaperones in combination with chaperones groEL-groES or only groEL-groES did not produce any soluble proteins. Baculovirus-insect expression of the aforementioned BTV(10)-VP2 recombinant proteins was carried out in Spodoptera frugiperda 9 (Sf9) cells. High level expression of the recombinant proteins was confirmed by an initial pilot expression study conducted at 42 hours post infection (p.i.). The pilot study also revealed that the recombinant proteins were insoluble. Arginine treatment of the lysate released a small fraction of soluble BTV(10)-VP2(aa450) and BTV(10)-VP2(ORF) proteins only detectable with immunoblot analysis using the anti-BTV(10) IgY antibodies. The amount of solubilized proteins was however too small to justify the cost associated with this expression system.Dissertation (MSc)--University of Pretoria, 2010.Geneticsunrestricte
Recommended from our members
New technologies accelerate the exploration of non-coding RNAs in horticultural plants.
Non-coding RNAs (ncRNAs), that is, RNAs not translated into proteins, are crucial regulators of a variety of biological processes in plants. While protein-encoding genes have been relatively well-annotated in sequenced genomes, accounting for a small portion of the genome space in plants, the universe of plant ncRNAs is rapidly expanding. Recent advances in experimental and computational technologies have generated a great momentum for discovery and functional characterization of ncRNAs. Here we summarize the classification and known biological functions of plant ncRNAs, review the application of next-generation sequencing (NGS) technology and ribosome profiling technology to ncRNA discovery in horticultural plants and discuss the application of new technologies, especially the new genome-editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, to functional characterization of plant ncRNAs
eCALIBRATOR : a comparative tool to identify key genes and pathways for eucalyptus defense against biotic stressors
Many pests and pathogens threaten Eucalyptus plantations. The study of defense responses in this economically important wood and fiber crop enables the discovery of novel pathways and genes, which may be adopted to improve resistance. Various functional genomics experiments have been conducted in Eucalyptus-biotic stress interactions following the availability of the Eucalyptus grandis genome, however, comparisons between these studies were limited largely due to a lack of comparative tools. To this end, we developed eCALIBRATOR http://ecalibrator.bi.up.ac.za, a tool for the comparison of Eucalyptus biotic stress interaction. The tool, which is not limited to Eucalyptus, allows the comparison of various datasets, provides a visual output in the form of Venn diagrams and clustering and extraction of lists for gene ontology enrichment analyses. We also demonstrate the usefulness of the tool in revealing pathways and key gene targets to further functionally characterize. We identified 708 differentially expressed E. grandis genes in common among responses to the insect pest Leptocybe invasa, oomycete pathogen Phytophthora cinnamomi and fungus Chrysoporthe austroafricana. Within this set of genes, one of the Gene Ontology terms enriched was “response to organonitrogen compound,” with NITRATE TRANSPORTER 2.5 (NRT2.5) being a key gene, up-regulated under susceptible interactions and downregulated under resistant interactions. Although previous functional genetics studies in Arabidopsis thaliana support a role in nitrate acquisition and remobilization under long-term nitrate starvation, the importance of NRT2.5 in plant defense is unclear. The T-DNA mutants of AtNRT2.5 were more resistant to Pseudomonas syringae pv. tomato pv tomato DC3000 inoculation than the wild-type counterpart, supporting a direct role for NRT2.5 in plant defense. Future studies will focus on characterizing the Eucalyptus ortholog of NRT2.5
eCALIBRATOR : a comparative tool to identify key genes and pathways for Eucalyptus defense against biotic stressors
Many pests and pathogens threaten Eucalyptus plantations. The study of defense
responses in this economically important wood and fiber crop enables the discovery
of novel pathways and genes, which may be adopted to improve resistance. Various
functional genomics experiments have been conducted in Eucalyptus-biotic stress
interactions following the availability of the Eucalyptus grandis genome, however,
comparisons between these studies were limited largely due to a lack of comparative
tools. To this end, we developed eCALIBRATOR http://ecalibrator.bi.up.ac.za, a tool for
the comparison of Eucalyptus biotic stress interaction. The tool, which is not limited
to Eucalyptus, allows the comparison of various datasets, provides a visual output in
the form of Venn diagrams and clustering and extraction of lists for gene ontology
enrichment analyses. We also demonstrate the usefulness of the tool in revealing
pathways and key gene targets to further functionally characterize. We identified 708
differentially expressed E. grandis genes in common among responses to the insect
pest Leptocybe invasa, oomycete pathogen Phytophthora cinnamomi and fungus
Chrysoporthe austroafricana. Within this set of genes, one of the Gene Ontology terms
enriched was “response to organonitrogen compound,” with NITRATE TRANSPORTER
2.5 (NRT2.5) being a key gene, up-regulated under susceptible interactions and downregulated
under resistant interactions. Although previous functional genetics studies in
Arabidopsis thaliana support a role in nitrate acquisition and remobilization under longterm
nitrate starvation, the importance of NRT2.5 in plant defense is unclear. The T-DNA mutants of AtNRT2.5 were more resistant to Pseudomonas syringae pv. tomato pv
tomato DC3000 inoculation than the wild-type counterpart, supporting a direct role
for NRT2.5 in plant defense. Future studies will focus on characterizing the Eucalyptus
ortholog of NRT2.5.Supplementary Material: Figure S1 : Selection of atnrt2.5 T-DNA mutants. (A) Diagram of the AtNRT2.5
gene consisting of three exons and two introns with the positions of the T-DNA
insertions in GK213H10 (AtNRT2.5-A) and GK046H04 (AtNRT2.5-B) in the
second and first exons, respectively. (B,C) PCR detection of T-DNA in
AtNRT2.5-A (B) and AtNRT2.5-B (C), respectively, using a combination of the
T-DNA left border oligonucleotide and gene-specific oligonucleotides. No
amplification was observed in AtNRT2.5-A and AtNRT2.5-B using gene-specific
oligonucleotides spanning the T-DNA insertion sites.Table S1 : Summary of the mapping of RNA-seq libraries per
Eucalyptus–pathogen interaction sample.Table S2 : Functionally enriched terms of the gene ontology biological processes
category in common between the resistant and susceptible defense responses.Table S3 : Functionally enriched terms of the gene ontology biological processes
category in the unique set of differentially expressed genes between the resistant
and susceptible interactions.The Department of Science and
Technology grant for Forest Genomics and Biotechnology,
the South African National Research Foundation Grant for
Y-rated researchers (UID105767) Incentive funding for rated
researchers (UID95807), Technology and Human Resources
for Industry Program (THRIP, Grant ID 96413) and the
Technology Innovation Agency (TIA) Forest Molecular Genetics
Cluster Program.http://www.frontiersin.org/Microbiologyam2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog
The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content
DUF1218 is a land plant-specific innovation and has previously been shown to be associated
with cell wall biology, vasculature patterning and abiotic/biotic stress response. The
Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are
preferentially expressed in the secondary cell wall depositing inflorescence stems. To further
our understanding of the roles of DUF1218-containing proteins in secondary cell wall
biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL
LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional
redundancy, we also characterized At4g19370 (MWL-2), the most closely related gene to
MWL-1 in the protein family. Subcellular localization revealed that both Arabidopsis proteins
are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2,
and independent overexpression lines showed no significant differences in plant growth or
changes in total lignin content relative to wild-type (WT) control plants. However, the double
homozygous mutant, mwl-1/mwl-2, had smaller rosettes with a significant decrease in
rosette fresh weight and stem height relative to the WT control at four weeks and six weeks,
respectively. Moreover, mwl-1/mwl-2 showed a significant reduction in total lignin content
(by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative
to the control plants. Our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin
biosynthesis, and these proteins appear to function redundantly.S1 Fig. Phylogenetic and bioinformatics analysis of all members of the Arabidopsis domain
of unknown function 1218 (DUF1218) family, and expression profiling of the candidate
members, MODIFYING WALL LIGNIN-1 (MWL-1, At1g31720) and MWL-2
(At4g19370). (A) Neighbor-joining phylogenetic tree of Arabdiopsis DUF1218-containing
proteins. ClustalW was used to align protein sequences from TAIR and the alignment thereafter
used to construct the tree using p-distance and pairwise deletion with 1000 bootstrap replicates
in MEGA5 [16]. Prediction of subcellular localization, signal peptide and number of
transmembrane domains was done using SUBA3 [31], Signal-3L [18] and TMHMM[19]
respectively, with default settings. Highlighted in pink are the related MWL-1 and 2 sequences.
(B) Arabidopsis expression profiles for MWL-1 and MWL-2 across different tissues during
development, exctracted from The Bio-Analytic Resource for Plant Biology (http://bar.
utoronto.ca/welcome.htm) [20]. Preferential expression is seen at distinct developmental
stages, however, there is overlap in the secondary cell wall depositing, 2nd internode region.
(DOCX)S2 Fig. Gene ontology enrichment of MWL-1 top 300 co-expressed genes in Arabidopsis.
Co-expressed genes were extracted from ATTED-II [10]. GO-full was conducted in Cytoscape
2.8.2 [22] using BiNGO 2.44 [21], while overrepresentation summary enrichment was performed
with the REVIGO server [23].
(DOCX)S3 Fig. Phenotypic analysis of At1g31720 (MWL-1) single T-DNA knockout line mutants
and MWL-1 overexpression lines. (A) RT-PCR detection of endogenous MWL-1 transcript in
the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative
RT-PCR analysis of MWL-1 overexpression lines 1 to 3 showing detection of MWL-1 transgene
in the transgenic lines. Actin2 was used as a control gene and RT-PCR was performed on
cDNA from stem tissue. Actin2 and MWL-1 gene-specific oligonucleotide sequences can be
found in S1 Table. Rosette size (C) and mass (D) of MWL-1 single T-DNA knockout line and
overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative (E) and quantitative
(F) stem length of MWL-1 single T-DNA knockout line and overexpression lines relative
to WT control at six weeks. For rosette mass n = 3 and for quantitative stem length n = 66.
Error bars indicate the standard error. Scale bar, 3 cm. Based on a two-tailed Student’s t-test
(P-value 0.05) no significant differences were seen in the growth and development of the single
mutant and transgenic OE lines in comparison to the WT controls.S4 Fig. Phenotypic analysis of At4g19370 (MWL-2) single T-DNA knockout line mutants
and MWL-2 overexpression lines. (A) RT-PCR detection of endogenous MWL-2 transcript in
the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative
RT-PCR analysis of MWL-2 overexpression lines 1 to 3 showing detection of MWL-2 transgene
in the transgenic lines except for OE1 which could be indicative of positional effect (position in
the genome), or co-suppression dominant repression. Actin2 was used as a control gene and
RT-PCR was performed on stem tissue. Actin2 and MWL-2 gene-specific oligonucleotide
sequences can be found in S1 Table. Rosette size (C) and mass (D) of MWL-2 single T-DNA
knockout line and overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative
(E) and quantitative (F) stem length of MWL-2 single T-DNA knockout line and overexpression
lines relative to WT control at six weeks. For rosette mass n = 3 and for quantitative
stem length n = 66. Error bars indicate the standard error while significant difference from the
WT based on a two-tailed Student’s t-test (P-value 0.05) is indicated by . Scale bar, 3 cm. No significant differences were seen in the growth and development of the single mutant and
transgenic OE lines in comparison to the WT controls except for OE-Line 2.
(DOCX)S5 Fig. Transverse sections of six-week-old stem tissue stained with phloroglucinol from
At1g31720 (MWL-1) and At4g19370 (MWL-2) T-DNA knockout mutant and overexpression
(OE) lines. Transverse sections from wildtype (WT) (A), At1g31720 mutant (B),
At4g19370 mutant (C), At1g31720 x At4g19370 double knockdown mutant (D), OEAt1g31720
line 1 (E), line 2 (F), line 3 (G), OEAt4g19370 line 1(H), line 2 (I), line 3 (J). Scale bar, 100ÎĽm
(indicated in red). No discernible differences were seen in the transverse sections of the single
and double mutant as well as the transgenic OE lines in comparison to the WT controls.
(DOCX)S1 Table. List of oligonucleotides used in the study.
(DOCX)S2 Table. Top 300 Arabidopsis co-expressed genes for MWL-1 (At1g31720) from ATTED-II
represented as MR value.
(DOCX)S3 Table. Top 300 Arabidopsis co-expressed genes for MWL-2 (At4g19370) from ATTED-II
represented as MR value.
(DOCX)S4 Table. Structural cell wall carbohydrates and lignin content from MWL-1 and MWL-2
overexpression, single and double knockout lines compared to its respective wildtype (WT)
control.
(DOCX)Sappi through the Forest Molecular Genetics
(FMG) Programme, the Technology and Human
Resources for Industry Programme (THRIP, UID
80118), and the National Research Foundation (NRF,
UID 71255 and 86936) of South Africa. RM
acknowledges an NRF Ph.D. Prestige and Equity
Scholarship.http://www.plosone.orgam2016Chemical EngineeringGenetic
Recombinant hyperthermophilic enzyme expression in plants : a novel approach for lignocellulose digestion
Plant biomass, as an abundant renewable carbon source,
is a promising alternative to fossil fuels. However, the
enzymes most commonly used for depolymerization of
lignocellulosic biomass are expensive, and the development
of cost-effective alternative conversion technologies
would be desirable. One possible option is the
heterologous expression of genes encoding lignocellulose-
digesting enzymes in plant tissues. To overcome
simultaneously issues of toxicity and incompatibility
with high-temperature steam explosion processes, the
use of heterologous genes encoding hyperthermophilic
enzymes may be an attractive alternative. This approach
could reduce the need for exogenous enzyme additions
prior to fermentation, reducing the cost of the complete
processing operation. This review highlights recent
advances and future prospects for using hyperthermophilic
enzymes in the biofuels industry.University of Pretoria and
National Research Foundation (NRF), South Africa.http://ac.els-cdn.com/hb201
Conservation and Diversification of Circadian Rhythmicity Between a Model Crassulacean Acid Metabolism Plant Kalanchoë fedtschenkoi and a Model C3 Photosynthesis Plant Arabidopsis thaliana
Crassulacean acid metabolism (CAM) improves photosynthetic efficiency under limited water availability relative to C3 photosynthesis. It is widely accepted that CAM plants have evolved from C3 plants and it is hypothesized that CAM is under the control of the internal circadian clock. However, the role that the circadian clock plays in the evolution of CAM is not well understood. To identify the molecular basis of circadian control over CAM evolution, rhythmic gene sets were identified in a CAM model plant species (Kalanchoë fedtschenkoi) and a C3 model plant species (Arabidopsis thaliana) through analysis of diel time-course gene expression data using multiple periodicity detection algorithms. Based on protein sequences, ortholog groups were constructed containing genes from each of these two species. The ortholog groups were categorized into five gene sets based on conservation and diversification of rhythmic gene expression. Interestingly, minimal functional overlap was observed when comparing the rhythmic gene sets of each species. Specifcally, metabolic processes were enriched in the gene set under circadian control in K. fedtschenkoi and numerous genes were found to have retained or gained rhythmic expression in K. fedtsechenkoi. Additonally, several rhythmic orthologs, including CAM-related orthologs, displayed phase shifts between species. Results of this analysis point to several mechanisms by which the circadian clock plays a role in the evolution of CAM. These genes provide a set of testable hypotheses for future experiments
The Kalanchoe genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism
Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops