Protein Body Biogenesis and Utility in Recombinant Protein Production in Nicotiana benthamiana

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles found in seeds whose function is to accumulate seed storage proteins. It was shown that PB formation is not limited to seeds, and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP), hydrophobin-I (HFBI) or Zera® fusion tags induces PBs in leaves of Nicotiana benthamiana. The mechanism by which fusion tags induce PBs is not well understood. To address how PBs form and develop in plant leaves, I studied the factors involved in their formation including recombinant protein concentration, effect of the fusion tags, PB sequestration patterns and destination in vivo. In this study I showed that PB formation is a concentration-dependent mechanism and that proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. The presence of fusion tags is not necessary for the formation of PBs but affects their distribution pattern and size. ELP-induced PBs are larger than HFBI-induced PBs and the size of both PBs increases over time along with accumulation levels of the recombinant protein. I found that in the process of PB formation, secretory and ER resident molecules are passively sequestered into the lumen of PBs. This property of PBs was proposed as a tool to increase accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins. To understand whether PBs are terminally-stored cytosolic organelles or if they are connected to the ER and to each other, I developed an EGFP-based photoconversion technique, which was successfully used to visualize the trafficking of proteins targeted to the cytosol, ER, apoplast, and chloroplast in vivo. Study of PBs with this technique suggested that PBs remain associated with the ER and communicate with one another via the ER

The SET Domain Protein, Set3p, Promotes the Reliable Execution of Cytokinesis in Schizosaccharomyces pombe

In response to perturbation of the cell division machinery fission yeast cells activate regulatory networks that ensure the faithful completion of cytokinesis. For instance, when cells are treated with drugs that impede constriction of the actomyosin ring (low doses of Latrunculin A, for example) these networks ensure that cytokinesis is complete before progression into the subsequent mitosis. Here, we identify three previously uncharacterized genes, hif2, set3, and snt1, whose deletion results in hyper-sensitivity to LatA treatment and in increased rates of cytokinesis failure. Interestingly, these genes are orthologous to TBL1X, MLL5, and NCOR2, human genes that encode components of a histone deacetylase complex with a known role in cytokinesis. Through co-immunoprecipitation experiments, localization studies, and phenotypic analysis of gene deletion mutants, we provide evidence for an orthologous complex in fission yeast. Furthermore, in light of the putative role of the complex in chromatin modification, together with our results demonstrating an increase in Set3p levels upon Latrunculin A treatment, global gene expression profiles were generated. While this analysis demonstrated that the expression of cytokinesis genes was not significantly affected in set3Δ backgrounds, it did reveal defects in the ability of the mutant to regulate genes with roles in the cellular response to stress. Taken together, these findings support the existence of a conserved, multi-protein complex with a role in promoting the successful completion of cytokinesis

Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

Enterohemorrhagic Escherichia coli (EHEC) has consistently been one of the foremost foodborne pathogen threats worldwide based on the past 30 years of surveillance. EHEC primarily colonizes the bovine gastrointestinal (GI) tract from which it can be transmitted to nearby farm environments and remain viable for months. There is an urgent need for effective and easily implemented pre-harvest interventions to curtail EHEC contamination of the food and water supply. In an effort to address this problem, we isolated single-domain antibodies (VHHs) specific for intimin, an EHEC adhesin required for colonization, and designed chimeric VHH fusions with secretory IgA functionality intended for passive immunotherapy at the mucosal GI surface. The antibodies were produced in leaves of Nicotiana benthamiana with production levels ranging between 1 and 3% of total soluble protein. in vivo assembly of all subunits into a hetero-multimeric complex was verified by co-immunoprecipitation. Analysis of multivalent protection across the most prevalent EHEC strains identified one candidate antibody, VHH10-IgA, that binds O145:Hnm, O111:Hnm, O26:H11, and O157:H7. Fluorometric and microscopic analysis also indicated that VHH10-IgA completely neutralizes the capacity of the latter three strains to adhere to epithelial cells in vitro. This study provides proof of concept that a plant-produced chimeric secretory IgA can confer cross-serotype inhibition of bacterial adhesion to epithelial cells

Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD). Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis

Identification of <i>S. pombe</i> genes differentially expressed with respect to genotype.

<p>Volcano plot (left panels) and scatter plot (right panels) analysis of the expression of all <i>S. pombe</i> genes in DMSO (<b>A</b>) or LatA (<b>B</b>) treated cells of the indicated genotype. Horizontal green lines in volcano plots represent a p-value of 0.05. Vertical green lines in volcano plots represent threshold for a 1.5 fold change in expression. Red squares in volcano plots indicate differentially expressed genes. Diagonal green lines in scatter plots represent the threshold for a 1.5 fold change in expression. The color of the squares in scatter plots indicates the level of expression of that gene in DMSO (<b>A</b>) or LatA (<b>B</b>) treated wild-type cells.</p

Domain structure of Set3p, Snt1p, and Hif2p.

<p>Structures are based upon Uniprot database predictions. Arabic numerals to the right of each schematic indicate the length of the protein in amino acids. Arabic numerals below each schematic indicate the amino acid position of that domain within the protein. Schematics are not drawn to scale.</p