Boosting in planta production of antigens derived from the porcine reproductive and respiratory syndrome virus (PRRSV) and subsequent evaluation of their immunogenicity

Porcine reproductive and respiratory syndrome (PRRS) is a disease of swine, caused by an arterivirus, the PRRS virus (PRRSV). This virus infects pigs worldwide and causes huge economic losses. Due to genetic drift, current vaccines are losing their power. Adaptable vaccines could provide a solution to this problem. This study aims at producing in planta a set of antigens derived from the PRRSV glycoproteins (GPs) to be included in a subunit vaccine. We selected the GP3, GP4 and GP5 and optimized these for production in an Arabidopsis seed platform by removing transmembrane domains (Tm) and/or adding stabilizing protein domains, such as the green fluorescent protein (GFP) and immunoglobulin (IgG) 'Fragment crystallizable' (Fc) chains. Accumulation of the GPs with and without Tm was low, reaching no more than 0.10% of total soluble protein (TSP) in homozygous seed. However, addition of stabilizing domains boosted accumulation up to a maximum of 2.74% of TSP when GFP was used, and albeit less effectively, also the Fc chains of the porcine IgG3 and murine IgG2a increased antigen accumulation, to 0.96% and 1.81% of TSP respectively, while the murine IgG3 Fc chain did not. Antigens with Tm were less susceptible to these manipulations to increase yield. All antigens were produced in the endoplasmic reticulum and accordingly, they carried high-mannose N-glycans. The immunogenicity of several of those antigens was assessed and we show that vaccination with purified antigens did elicit the production of antibodies with virus neutralizing activity in mice but not in pigs

A Monomer-to-Trimer Transition of the Human Mitochondrial Transcription Termination Factor (mTERF) Is Associated with a Loss of in Vitro Activity

The human mitochondrial transcription termination factor (mTERF) is a nuclear-encoded 39-kDa protein that recognizes a mtDNA segment within the mitochondrial tRNALeu(UUR) gene immediately adjacent to and downstream of the 16 S rRNA gene. Binding of mTERF to this site promotes termination of rDNA transcription. Despite the fact that mTERF binds DNA as a monomer, the presence in its sequence of three leucine-zipper motifs suggested the possibility of mTERF establishing intermolecular interactions with proteins of the same or different type. When a mitochondrial lysate from HeLa cells was submitted to gel filtration chromatography, mTERF was eluted in two peaks, as detected by immunoblotting. The first peak, which varied in proportion between 30 and 50%, appeared at the position expected from the molecular mass of the monomer (41 ± 2 kDa), and the gel filtration fractions that contained it exhibited DNA binding activity. Most interestingly, the material in this peak had a strong stimulating activity on in vitro transcription of the mitochondrial rDNA. The second peak eluted at a position corresponding to an estimated molecular mass of 111 ± 5 kDa. No mTERF DNA binding activity could be detected in the corresponding gel filtration fractions. Therefore, we propose that mTERF exists in mitochondria in two forms, an active monomer and an inactive large size complex. The estimated molecular weight of this complex and the fact that purified mTERF can be eluted from a gel filtration column as a complex of the same molecular weight strongly suggest that this inactive complex is a homotrimer of mTERF

Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus

A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-ß-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to >90% homogeneity by affinity chromatography on p-aminophenyl-ß-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-ß-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-ß-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B

In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development

Acknowledgments: The authors would like to acknowledge the funding support for this work from Scottish Enterprise [VNAR_001(2012)] and the Biotechnology and Biological Sciences Research Council (BB/K010905/1).Peer reviewedPublisher PD

Structural basis for sequence specific DNA binding and protein dimerization of HOXA13.

The homeobox gene (HOXA13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of genes during embryonic morphogenesis. Here we present the NMR structure of HOXA13 homeodomain (A13DBD) bound to an 11-mer DNA duplex. A13DBD forms a dimer that binds to DNA with a dissociation constant of 7.5 nM. The A13DBD/DNA complex has a molar mass of 35 kDa consistent with two molecules of DNA bound at both ends of the A13DBD dimer. A13DBD contains an N-terminal arm (residues 324 - 329) that binds in the DNA minor groove, and a C-terminal helix (residues 362 - 382) that contacts the ATAA nucleotide sequence in the major groove. The N370 side-chain forms hydrogen bonds with the purine base of A5* (base paired with T5). Side-chain methyl groups of V373 form hydrophobic contacts with the pyrimidine methyl groups of T5, T6* and T7*, responsible for recognition of TAA in the DNA core. I366 makes similar methyl contacts with T3* and T4*. Mutants (I366A, N370A and V373G) all have decreased DNA binding and transcriptional activity. Exposed protein residues (R337, K343, and F344) make intermolecular contacts at the protein dimer interface. The mutation F344A weakens protein dimerization and lowers transcriptional activity by 76%. We conclude that the non-conserved residue, V373 is critical for structurally recognizing TAA in the major groove, and that HOXA13 dimerization is required to activate transcription of target genes

Advances in DNA Affinity Chromatography

Different aspects of DNA affinity chromatography such as DNA complexity heparin elution, the Bi-column method and the oligonucluotide trapping method were studied. The complexity (length) of a DNA sequence attached to an affinity chromatography column affects column retention, and the purity of transcription factors obtained. T18: A18 tailed DNA affinity columns were better suited for purification of most of the transcription factors than either the discrete or concatemeric DNA affinity columns. A novel method using heparin for eluting transcription factors from DNA Sepharose columns was characterized. The amount of the lac repressor chimera which eluted from the column was shown to increase with increases in the mobile phase heparin concentration. The elution of the protein was also shown to be dependent on the amount of DNA coupled to the column and more protein eluted from columns containing lesser amounts of DNA. These data suggest that heparin and DNA compete for binding to the protein; this competition causes elution. Comparison of heparin- and salt-eluted protein demonstrated the heparin-eluted fraction of lac repressor was significantly purer than that eluted with salt and comparable to that obtained by elution with the specific ligand IPTG, a lactose analog. A novel Bi-column method was developed in which lac repressor is eluted from the Op1-Sepharose with a low heparin concentration and trapped on a Op1T18-Sepharose column because of its higher affinity for the lac repressor protein. Elution of the latter column with buffer containing a high salt concentration gives significantly purer transcription factor than the conventionally used single column methods and removes residual heparin. Highly pure CAAT enhancer binding protein(C/EBP) and the B3 transcription factor are also obtained by using variants of this Bi-column method. A new oligonucleotide trapping method in which a short oligonucleotide coupled to Sepharose is used to trap a complex of the transcription factor and its corresponding specific DNA sequence was developed. Highly purified transcription factor B3 was obtained using the oligonucleotide trapping method

The hepta-β-glucoside elicitor-binding proteins from legumes represent a putative receptor family

The ability of legumes to recognize and respond to β-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound β-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-β-glucoside conjugate (K-d = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (K-d = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex

Site directed mutagenesis and purification of the cDNA for human class I aldehyde dehydrogenase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University

Aldehyde dehydrogenase (ALDH) is a key enzyme of alcohol metabolism, removing acetaldehyde which is formed as a product of the alcohol dehydrogenase reaction. If acetaldehyde is not effectively removed, acetaldehyde accumulates and produces an adverse reaction to alcohol, with nausea, flushing and increased heart rate and blood pressure. ALDH is involved in the conversion of retinal to retinoic acid (RA). RA has recently been shown to bind to receptors, which then act as nuclear transcription factors and play important roles in foetal development and maintenance of the epithelial layer in the body. Interference by ethanol and perhaps by acetaldehyde with this process is probably the cause of Foetal Alcohol Syndrome. In addition ALDH is also involved in the metabolism of catecholamine neurotransmitters, plays a role in the removal of toxic substances from the body and may have a role in protection against some chemical carcinogens. Dr. Kerrie Jones had obtained moderate levels of expression of recombinant ALDH in E. coli and constructed a number of mutants chosen on the basis of chemical modification data and sequence alignment. Mutant proteins were also expressed and assayed for enzyme activity in crude extracts. The aim of this thesis was to improve purification and yield of the expressed ALDH proteins. By the use of site-directed mutagenesis I attempted to mutate the amino acid residue Lys272 to either alanine, histidine or arginine. Future comparison of the properties of the site-directed mutants with those of the wild type enzyme will help to determine the importance of the residue (which has been replaced by mutagenesis) to catalysis