Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii.

Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a "plant-like" algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN

Inner/Outer Nuclear Membrane Fusion in Nuclear Pore Assembly: Biochemical Demonstration and Molecular Analysis

The nuclear pore complex (NPC) is characterized by a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel, within which the nuclear pore is built, has little evolutionary precedent. In this report we demonstrate and map the inner/outer nuclear membrane fusion in NPC assembly

Defining the early steps in nuclear pore assembly : chromatin-associated ELYS initiates pore assembly

My thesis is focused upon molecularly defining the mechanism of early steps in metazoan nuclear pore assembly. Nuclear pore complexes (NPCs) are large proteinaceous structures that span the nuclear envelope and act as gated aqueous channels to regulate the transport of macromolecules between the nucleus and cytoplasm. In metazoans, the massive NPCs disassemble into soluble subunits at the beginning of mitosis and then somehow reassemble following chromosome segregation; a process that is coordinated with membrane recruitment and fusion. The mechanism and order of NPC assembly is poorly understood. In Chapter 1, I show that ELYS co-purifies with the Nup107-160 complex, the largest subunit of the NPC, in Xenopus egg and human cell extracts. Indeed, I demonstrate that ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division. In Chapter 2, I focus on defining the early steps in nuclear pore assembly, including the mechanism for ELYS as the pore 'targeting' protein. In Chapter 3, I collaborated with Dr. Corinne Ramos on a study to order pore assembly with respect to inner and outer nuclear membrane fusion. Taken the data from chapters 1-3 together, I suggest a model in which NPC assembly is initiated on AT -rich chromatin through an interaction with the C-terminus of ELYS. The data also show the chromatin binding of ELYS precedes and is required for the binding of the Nup107-160 complex. Chromatin-bound ELYS and the Nup107-160 complex then recruit integral pore membrane proteins POM121- and NDC1-containing membrane vesicles. Membrane vesicle fusion takes place to form patches of continuous double nuclear membranes. Oligomerization of ELYS/Nup107-160/POM121 then acts to promote fusion between the inner and outer nuclear membranes to form a diffusion channel. Finally, the remaining soluble pore subunits are recruited to assemble the mature, functional nuclear pore. Finally, Appendix B presents data on the Xenopus binding partners of the C- terminus of Nup160, a member of the Nup107-160 complex, which was derived from mass spectrometry analyses. This data led to the discovery that vertebrate centrin 2 localizes to NPCs and functions in mRNA and protein export, as described in Chapter

Centrin 2 Localizes to the Vertebrate Nuclear Pore and Plays a Role in mRNA and Protein Export▿ †

Centrins in vertebrates have traditionally been associated with microtubule-nucleating centers such as the centrosome. Unexpectedly, we found centrin 2 to associate biochemically with nucleoporins, including the Xenopus laevis Nup107-160 complex, a critical subunit of the vertebrate nuclear pore in interphase and of the kinetochores and spindle poles in mitosis. Immunofluorescence of Xenopus cells and in vitro reconstituted nuclei indeed revealed centrin 2 localized at the nuclear pores. Use of the mild detergent digitonin in immunofluorescence also allowed centrin 2 to be clearly visualized at the nuclear pores of human cells. Disruption of nuclear pores using RNA interference of the pore assembly protein ELYS/MEL-28 resulted in a specific decrease of centrin 2 at the nuclear rim of HeLa cells. Functionally, excess expression of either the N- or C-terminal calcium-binding domains of human centrin 2 caused a dominant-negative effect on both mRNA and protein export, leaving protein import intact. The mRNA effect mirrors that found for the Saccharomyes cerevisiae centrin Cdc31p at the yeast nuclear pore, a role until now thought to be unique to yeast. We conclude that in vertebrates, centrin 2 interacts with major subunits of the nuclear pore, exhibits nuclear pore localization, and plays a functional role in multiple nuclear export pathways

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii.

Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a "plant-like" algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN

Enhanced genetic tools for engineering multigene traits into green algae.

Transgenic microalgae have the potential to impact many diverse biotechnological industries including energy, human and animal nutrition, pharmaceuticals, health and beauty, and specialty chemicals. However, major obstacles to sophisticated genetic and metabolic engineering in algae have been the lack of well-characterized transformation vectors to direct engineered gene products to specific subcellular locations, and the inability to robustly express multiple nuclear-encoded transgenes within a single cell. Here we validate a set of genetic tools that enable protein targeting to distinct subcellular locations, and present two complementary methods for multigene engineering in the eukaryotic green microalga Chlamydomonas reinhardtii. The tools described here will enable advanced metabolic and genetic engineering to promote microalgae biotechnology and product commercialization

Enhanced Genetic Tools for Engineering Multigene Traits into Green Algae

Transgenic microalgae have the potential to impact many diverse biotechnological industries including energy, human and animal nutrition, pharmaceuticals, health and beauty, and specialty chemicals. However, major obstacles to sophisticated genetic and metabolic engineering in algae have been the lack of well-characterized transformation vectors to direct engineered gene products to specific subcellular locations, and the inability to robustly express multiple nuclear-encoded transgenes within a single cell. Here we validate a set of genetic tools that enable protein targeting to distinct subcellular locations, and present two complementary methods for multigene engineering in the eukaryotic green microalga Chlamydomonas reinhardtii. The tools described here will enable advanced metabolic and genetic engineering to promote microalgae biotechnology and product commercialization

Aminooxypentane addition to the chemokine macrophage inflammatory Protein-1 alpha P increases receptor affinities and HIV inhibition

To enter its target cells, human immunodeficiency virus (HIV) must interact with CD4 and one of a family of chemokine receptors. CCR5 is widely used by the virus in this context, and its ligands can prevent HIV entry. Amino-terminal modified chemokine variants, in particular AOP-RANTES (aminooxypentane-linked regulated on activation normal T cell expressed and secreted), exhibit enhanced HIV entry inhibition. We have previously demonstrated that a non-allelic isoform of macrophage inflammatory protein (MIP)-1α, termed MIP-1αP, is the most active naturally occurring inhibitor of HIV entry known. Here we report the properties of a variant of MIP-1αP with an AOP group on the amino terminus. We show that, like RANTES, the addition of AOP to MIP-1αP enhances its interactions with CCR1 and CCR5, allows more effective internalization of CCR5, and increases the ligand's potency as an inhibitor of HIV entry through CCR5. Importantly, AOP-MIP-1αP is about 10-fold more active than AOP-RANTES at inhibiting HIV entry, making it the most effective chemokine-based inhibitor of HIV entry through CCR5 described to date. Surprisingly, the enhanced receptor interactions of AOP-MIP-1αP do not translate into increased chemotaxis or coupling to calcium ion fluxes, suggesting that this protein should be viewed as a partial, rather than a full, agonist for CCR1 and CCR5

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii

Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN