CA2555137A1 - Puroindolines as fusion-protein carriers in molecular pharming - Google Patents
Puroindolines as fusion-protein carriers in molecular pharming Download PDFInfo
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Abstract
Described are methods and compositions for producing proteinated granules and isolating desired products (for example, one or more protein, peptide, remediant, cosmeceutical, or pharmaceutical compound species) in transgenic plants engineered to produce the desired product(s). The present invention provides polynucleotide and polypeptide and protein sequences from plants to serve as carriers of recombinant fusion "cargo" proteins (also called "hybrid" or "
multimeric" polypeptides), methods for targeting fusion polypeptides to starch granules, methods for tethering fusion polypeptides to the lipid layer associated with starch granule surfaces, and methods for using starch granule associated polypeptides as a carrier protein for the production of recombinant biopharmaceuticals. The desired products are isolated from the transgenic plants by size reduction (also called "milling") and air classification to remove the proteinated granule away from the endogenous host proteome.
multimeric" polypeptides), methods for targeting fusion polypeptides to starch granules, methods for tethering fusion polypeptides to the lipid layer associated with starch granule surfaces, and methods for using starch granule associated polypeptides as a carrier protein for the production of recombinant biopharmaceuticals. The desired products are isolated from the transgenic plants by size reduction (also called "milling") and air classification to remove the proteinated granule away from the endogenous host proteome.
Description
Altosaar 18022 BACKGROUND Strategic Proposal The current proposal represents a natural confluence of two major research thrusts in our laboratory: the genetic modification of plants for the production of economically important proteins, and the study of cereal seed proteins synthesis, translocation and deposition (Blais, 2006).
Production of plant-made pharmaceuticals In the area of recombinant proteins (biopharmaceuticals), Plant-made phannaceutical (PMP) proteins are fast becoming targets of commercialization (Ma, 2005). The advantages which plants offer in terms of production scalability, cost, product safety, ease of storage, and distribution cannot be matched by any current commercial system. The first transgenic plants, reported in 1983, were created via Agrobacterium-mediated transformation to confer kanamycin resistance (Zambryski, 1983). The first PMP, human serum albumin, was produced in 1990 in transgenic potato and tobacco (Sijmons, 1990).
Since then, greater than 100 unique peptides have been produced in plants using recombinant DNA
technology (Twyman, 2003) and about a thousand medical and non-medical candidate proteins have been identified to date for Molecular Farming (Marshall, 2006). Today, new plant-derived pharmaceuticals used to treat human disease are reported almost weekly.
Several human PMPs have reached late stages of commercial development. These include gastric lipase, a therapeutic enzyme for treating cystic fibrosis and pancreatitis, and Human Intrinsic Factor, a dietary supplement to stem vitamin B 12 deficiency (Ma, 2005). Others are still awaiting approval for clinical trials on humans (Ma, 2005). Considering also veterinary applications, a milestone was achieved in January 2006 when the USDA Center for Veterinary Biologics issued regulatory approval to Dow AgroSciences for its Newcastle Disease Virus vaccine for poultry (http=//www dowagro com/newsroom/corporatenews/2006/20060 1 3 1b.htm viewed 18.4.06).
Plant-based molecular farming has been consistently validated as a feasible technology and proof-of-concept has been demonstrated by several companies including for example Calgary's SemBioSys Genetics Inc. (seed oil body as carrier) and Quebec's MedicaGo (alfalfa foliage fractionation). While most plant-based molecular farming technologies share similar beneficial characteristics, there are many technical hurdles and development choices to be made and this technology is not without its shortcomings; most notable being yield, enrichment of active protein, and strategies for downstream processing. This Strategic project proposes a method to enhance the accumulation of recombinant protein in cereal seed storage organelles for the quick and cost-effective purification of pharmaceutically active peptides.
Currently, most pharmaceutically active proteins are produced in bacterial and animal cell cultures or in whole animals. The cost of maintaining cell lines and highly qualified personnel is greater than maintaining plants. It is estimated that the cost of producing pharmaceutically active proteins in plants is less than one percent of producing them in mammal cell cultures and
Production of plant-made pharmaceuticals In the area of recombinant proteins (biopharmaceuticals), Plant-made phannaceutical (PMP) proteins are fast becoming targets of commercialization (Ma, 2005). The advantages which plants offer in terms of production scalability, cost, product safety, ease of storage, and distribution cannot be matched by any current commercial system. The first transgenic plants, reported in 1983, were created via Agrobacterium-mediated transformation to confer kanamycin resistance (Zambryski, 1983). The first PMP, human serum albumin, was produced in 1990 in transgenic potato and tobacco (Sijmons, 1990).
Since then, greater than 100 unique peptides have been produced in plants using recombinant DNA
technology (Twyman, 2003) and about a thousand medical and non-medical candidate proteins have been identified to date for Molecular Farming (Marshall, 2006). Today, new plant-derived pharmaceuticals used to treat human disease are reported almost weekly.
Several human PMPs have reached late stages of commercial development. These include gastric lipase, a therapeutic enzyme for treating cystic fibrosis and pancreatitis, and Human Intrinsic Factor, a dietary supplement to stem vitamin B 12 deficiency (Ma, 2005). Others are still awaiting approval for clinical trials on humans (Ma, 2005). Considering also veterinary applications, a milestone was achieved in January 2006 when the USDA Center for Veterinary Biologics issued regulatory approval to Dow AgroSciences for its Newcastle Disease Virus vaccine for poultry (http=//www dowagro com/newsroom/corporatenews/2006/20060 1 3 1b.htm viewed 18.4.06).
Plant-based molecular farming has been consistently validated as a feasible technology and proof-of-concept has been demonstrated by several companies including for example Calgary's SemBioSys Genetics Inc. (seed oil body as carrier) and Quebec's MedicaGo (alfalfa foliage fractionation). While most plant-based molecular farming technologies share similar beneficial characteristics, there are many technical hurdles and development choices to be made and this technology is not without its shortcomings; most notable being yield, enrichment of active protein, and strategies for downstream processing. This Strategic project proposes a method to enhance the accumulation of recombinant protein in cereal seed storage organelles for the quick and cost-effective purification of pharmaceutically active peptides.
Currently, most pharmaceutically active proteins are produced in bacterial and animal cell cultures or in whole animals. The cost of maintaining cell lines and highly qualified personnel is greater than maintaining plants. It is estimated that the cost of producing pharmaceutically active proteins in plants is less than one percent of producing them in mammal cell cultures and
2-10% the cost of using microbial fermentation systems for the same task (Kusnadi, 1997). Related to development costs is scalability. Fermentation systems and transgenic animals have limited potential in this respect, whereas the scale of plant-based production can be quickly modified in response to market demand by using more or less land as needed. The speed of scale-up is also important. It can take several years to achieve a ten fold scale-up in a herd of transgenic sheep using natural breeding cycles, while transgenic plants can be scaled-up more than one thousand fold in one generation due to the prolific nature of seeds (Schillberg, 2002).
Animal cell cultures have the advantage of compatible post-translational modifications for animal-derived peptides and proteins that might otherwise be left unmodified or modified incorrectly if produced in bacteria. However, the use of animal cell cultures, especially mammalian cell cultures, brings up the possibility of disease-causing organisms being transmitted from culture to end-user. A
Altosaar 18022 major advantage of plant-based systems is the lack of disease-causing organisms that can pass from plants to humans.
The molecular mechanisms that drive the expression of transgenes and the subsequent localization and modification of recombinant proteins in genetically modified plants are not fully understood. The percentage of recombinant protein that will be obtained can only be determined empirically and is known to vary greatly, anywhere from 0.0026% soluble leaf protein for human Erythropoietin expressed in tobacco to 14.4% soluble leaf protein for a fungal phytase expressed in tobacco (Hellwig, 2004). Despite this necessity for a case-by-case approach, strategies do exist to increase expression, and enrichment of recombinant proteins, allowing for less cumbersome extraction methods. Techniques to improve expression have included choosing strong, organ-specific or ubiquitous promoters and codon optimization of reading frames (Cheng, 1999; Blais, 2006).
Further improvements in overall yield have been sought using localization strategies. One such tactic is the expression of the particular recombinant protein of interest in the seeds of transgenic cereal plants (Hood, 1997). High levels of the product can accumulate in a small volume, which minimizes the costs associated with processing (Stoger, 2000). Often, recombinant proteins will accumulate in naturally occurring protein storage compartments known as protein bodies. For example, in our hands, human cytomegalovirus coat protein hCMV gB, as a vaccine candidate, appears to have cryptic targeting signals because gold immunolocalization detected it mostly in seed protein bodies (Wright, 2001).
Downstream processing of seeds with protein purification methods can then be tailored knowing that recombinant protein has been enriched in these areas. In another example, oil bodies in oilseed crops have been used as sites of rt-protein deposition. Desirable proteins expressed as a fusion with oleosin will localize to oil bodies in these plants, simplifying the subsequent purification steps which follow seed crushing and oil extraction (Kuhnel, 2003).
Besides seeds, PMP production platforms include total foliage, leaf juice, leaf interstitial spaces, cane sap, latex, banana fruit, potato, algae... Direct consumption (oral delivery) is an option but most regulators require purification as rt-protein dosage is difficult to maintain due to variation of fruit development. Thus, up to 85% of manufacturing costs occur due to downstream processing. Developing a generally applicable, economic, large-scale purification strategy becomes desirable (Ma, 2005).
Choosing the appropriate plant, organ, tissue, and sub-cellular organelle and/or compartment to be targeted by a pharmaceutically active peptide would be a great boon for planning a manufacturing process. It would give greater reliability to predicting yields and would greatly decrease downstream processing costs. Our lab studies seeds because of their renowned stability for protecting expensive Pharma proteins in a dry, stable, active conformation. We propose to take advantage of the starch granule surface in cereal seeds as a storage compartment for producing pharmaceutically active proteins in cereal crops.
Starch Granule-associated proteins - Puroindolines/Tryptophanins I have been working on cereal seed protein chemistry for over 25 years.
Recently, this work has focused on the puroindoline/tryptophanin family of seed-specific proteins -the puroindolines in Triticum sp. and the tryptophanins (avenoindolines) in oats (A. sativa).
Puroindolines (PINs) are low molecular weight, endosperm-specific, highly surface active, cysteine-rich, basic, amphiphilic, non-gluten, lipid-binding proteins that were originally isolated from bread wheat (Triticum aestivum L. em Thell.) kernels using Triton X-1 14 phase partitioning (Blochet, 1993). Two distinct puroindolines have been identified; a major isoform, called puroindoline-a (PIN-a) which has a unique tryptophan-rich sequence motif (-Trp-Arg-Trp-Trp-Lys-Trp-Trp-Lys-), and, a minor isoform, puroindoline-b (PIN-b) with a truncated motif (-Trp-Pro-Thr-Trp-Trp-Lys-) (Gautier, 1994).
As reviewed in detail by Morris (2002), PINs constitute the primary components of "friabilin"
andpin genes have been linked to the friabilin genes and the "Hardness" (Ha) locus on the short arm of chromosome 5D (5DS) in wheat. More recently, expression ofpin genes in rice has led to enhanced Altosaar 18022 grain softness (Krishnamurthy and Giroux, 2001). Taken in totality the available evidence indicates that PINs form the molecular basis of wheat grain hardness (softness) or texture (Morris 2002).
The available evidence (reviewed by Morris, 2002; Turnbull and Rahman, 2002) is consistent with the following mechanism for grain softness. PINs initially accumulate in the protein bodies of the -? ~ ~ : = : .. . : . . . : developing wheat grain. As the seed matures, these protein bodies 'Hera ePaa8pe~m ieXi~re ~J. disintegrate resulting in the formation of a protein matrix in which starch granules become embedded. At this point, a portion of the total PIN proteins is retranslocated and becomes associated with the surface ~j: :... ..::: .. ' of starch granules, possibly through binding to lipids in amyloplast A dhesion' between gianule =, ~ membrane remnants on the surface of the starch granules. Hard and soft : surtece and p.oAein. metriX :=. wheats differ in the amount of starch-associated PINs with soft wheat soft eriaogpe:m, i:Xcu.e '... having significantly larger amounts of starch-bound PINs. It has been proposed (Schofield and Greenwell, 1987 - see Fig.) that the starch-bound PINs act as "non-stick" proteins allowing soft wheat to be milled into flour using lower amounts of force and with fractures occurring at the starch granule/protein matrix interface.
Pres.noe of I sk 'peaieih' PIN proteins may also play a role in protecting wheat seeds interferes with adhesion against against fungal infections (Blochet, 1993). PIN proteins exhibited a synergistic inhibitory effect against fungal growth when mixed with other antimicrobial proteins such as purothionins. In addition, constitutive expression of pin genes in rice led to a significant increase in tolerance to rice blast infection caused by Magnaporthe grisea and reduced in vitro growth of both M. grisea and Rhizoctonia solani (sheath blight), two major debilitating rice pathogens (Krishnamurthy, 2001). Like thionins and nsLTPs, PIN proteins have a high affinity for tight binding to polar lipids, a mechanism that could be responsible for their fmal deposition on the starch granule, and their membranotoxic effects on fungal pathogens.
Work in our lab (Tanchak, 1998) and by other groups (reviewed by Morris, 2002) has identified and characterized puroindoline-like genes, cDNAs and/or polypeptides in a variety of other agriculturally important monocotyledonous crop species such as barley (Hordeum vulgare L.), oat (Avena sativa L.), rye (Secale cerealis L.). [We named the proteins in oats tryptophanin (Tanchak, 1998), also known as avenoindolines.] In contrast, other major monocotyledonous crops such corn (Zea mays L.), rice (Oryza sativa L.) and pasta wheat (Triticum durum L.) do not possess puroindoline-like sequences. [Recent evidence suggests that rice possesses a gene related to the PINs of wheat. It is believed that this rice sequence was not detected in earlier work as the degree of similarity is below the threshold of detection attainable with the hybridization conditions used in the earlier studies (Chantret, 2004).]
Other recent advances in our study of oat tryptophanins include the results of Triton X-114 phase-partitioning (Tanchak, 1998) and the immunolocalization of puroindoline-like proteins in oats seeds (Melnyk, manuscript in preparation). The phase-partitioning experiments show the presence of a major 14 kDa polypeptide in the detergent-rich phase. N-terminal sequencing of this polypeptide confirms that it is the mature polypeptide encoded by one of the oat tryptophanin cDNA clones isolated in our lab. Immunolocalization, using a monoclonal antibody preparation specific for "friabilin" (aka puroindoline), shows labeling on the surface of starch granules in the endosperm of oat seeds (MAb courtesy of Rhone Diagnostics, Glasgow). Although the exact specificity of this monoclonal preparation is not known (i.e. PIN-a and/or PIN-b), these results suggest that the relevant proteins in oats behave in a similar manner as the puroindolines in wheat. Because of the strong homology among the wild-type proteins from oat and wheat:
Animal cell cultures have the advantage of compatible post-translational modifications for animal-derived peptides and proteins that might otherwise be left unmodified or modified incorrectly if produced in bacteria. However, the use of animal cell cultures, especially mammalian cell cultures, brings up the possibility of disease-causing organisms being transmitted from culture to end-user. A
Altosaar 18022 major advantage of plant-based systems is the lack of disease-causing organisms that can pass from plants to humans.
The molecular mechanisms that drive the expression of transgenes and the subsequent localization and modification of recombinant proteins in genetically modified plants are not fully understood. The percentage of recombinant protein that will be obtained can only be determined empirically and is known to vary greatly, anywhere from 0.0026% soluble leaf protein for human Erythropoietin expressed in tobacco to 14.4% soluble leaf protein for a fungal phytase expressed in tobacco (Hellwig, 2004). Despite this necessity for a case-by-case approach, strategies do exist to increase expression, and enrichment of recombinant proteins, allowing for less cumbersome extraction methods. Techniques to improve expression have included choosing strong, organ-specific or ubiquitous promoters and codon optimization of reading frames (Cheng, 1999; Blais, 2006).
Further improvements in overall yield have been sought using localization strategies. One such tactic is the expression of the particular recombinant protein of interest in the seeds of transgenic cereal plants (Hood, 1997). High levels of the product can accumulate in a small volume, which minimizes the costs associated with processing (Stoger, 2000). Often, recombinant proteins will accumulate in naturally occurring protein storage compartments known as protein bodies. For example, in our hands, human cytomegalovirus coat protein hCMV gB, as a vaccine candidate, appears to have cryptic targeting signals because gold immunolocalization detected it mostly in seed protein bodies (Wright, 2001).
Downstream processing of seeds with protein purification methods can then be tailored knowing that recombinant protein has been enriched in these areas. In another example, oil bodies in oilseed crops have been used as sites of rt-protein deposition. Desirable proteins expressed as a fusion with oleosin will localize to oil bodies in these plants, simplifying the subsequent purification steps which follow seed crushing and oil extraction (Kuhnel, 2003).
Besides seeds, PMP production platforms include total foliage, leaf juice, leaf interstitial spaces, cane sap, latex, banana fruit, potato, algae... Direct consumption (oral delivery) is an option but most regulators require purification as rt-protein dosage is difficult to maintain due to variation of fruit development. Thus, up to 85% of manufacturing costs occur due to downstream processing. Developing a generally applicable, economic, large-scale purification strategy becomes desirable (Ma, 2005).
Choosing the appropriate plant, organ, tissue, and sub-cellular organelle and/or compartment to be targeted by a pharmaceutically active peptide would be a great boon for planning a manufacturing process. It would give greater reliability to predicting yields and would greatly decrease downstream processing costs. Our lab studies seeds because of their renowned stability for protecting expensive Pharma proteins in a dry, stable, active conformation. We propose to take advantage of the starch granule surface in cereal seeds as a storage compartment for producing pharmaceutically active proteins in cereal crops.
Starch Granule-associated proteins - Puroindolines/Tryptophanins I have been working on cereal seed protein chemistry for over 25 years.
Recently, this work has focused on the puroindoline/tryptophanin family of seed-specific proteins -the puroindolines in Triticum sp. and the tryptophanins (avenoindolines) in oats (A. sativa).
Puroindolines (PINs) are low molecular weight, endosperm-specific, highly surface active, cysteine-rich, basic, amphiphilic, non-gluten, lipid-binding proteins that were originally isolated from bread wheat (Triticum aestivum L. em Thell.) kernels using Triton X-1 14 phase partitioning (Blochet, 1993). Two distinct puroindolines have been identified; a major isoform, called puroindoline-a (PIN-a) which has a unique tryptophan-rich sequence motif (-Trp-Arg-Trp-Trp-Lys-Trp-Trp-Lys-), and, a minor isoform, puroindoline-b (PIN-b) with a truncated motif (-Trp-Pro-Thr-Trp-Trp-Lys-) (Gautier, 1994).
As reviewed in detail by Morris (2002), PINs constitute the primary components of "friabilin"
andpin genes have been linked to the friabilin genes and the "Hardness" (Ha) locus on the short arm of chromosome 5D (5DS) in wheat. More recently, expression ofpin genes in rice has led to enhanced Altosaar 18022 grain softness (Krishnamurthy and Giroux, 2001). Taken in totality the available evidence indicates that PINs form the molecular basis of wheat grain hardness (softness) or texture (Morris 2002).
The available evidence (reviewed by Morris, 2002; Turnbull and Rahman, 2002) is consistent with the following mechanism for grain softness. PINs initially accumulate in the protein bodies of the -? ~ ~ : = : .. . : . . . : developing wheat grain. As the seed matures, these protein bodies 'Hera ePaa8pe~m ieXi~re ~J. disintegrate resulting in the formation of a protein matrix in which starch granules become embedded. At this point, a portion of the total PIN proteins is retranslocated and becomes associated with the surface ~j: :... ..::: .. ' of starch granules, possibly through binding to lipids in amyloplast A dhesion' between gianule =, ~ membrane remnants on the surface of the starch granules. Hard and soft : surtece and p.oAein. metriX :=. wheats differ in the amount of starch-associated PINs with soft wheat soft eriaogpe:m, i:Xcu.e '... having significantly larger amounts of starch-bound PINs. It has been proposed (Schofield and Greenwell, 1987 - see Fig.) that the starch-bound PINs act as "non-stick" proteins allowing soft wheat to be milled into flour using lower amounts of force and with fractures occurring at the starch granule/protein matrix interface.
Pres.noe of I sk 'peaieih' PIN proteins may also play a role in protecting wheat seeds interferes with adhesion against against fungal infections (Blochet, 1993). PIN proteins exhibited a synergistic inhibitory effect against fungal growth when mixed with other antimicrobial proteins such as purothionins. In addition, constitutive expression of pin genes in rice led to a significant increase in tolerance to rice blast infection caused by Magnaporthe grisea and reduced in vitro growth of both M. grisea and Rhizoctonia solani (sheath blight), two major debilitating rice pathogens (Krishnamurthy, 2001). Like thionins and nsLTPs, PIN proteins have a high affinity for tight binding to polar lipids, a mechanism that could be responsible for their fmal deposition on the starch granule, and their membranotoxic effects on fungal pathogens.
Work in our lab (Tanchak, 1998) and by other groups (reviewed by Morris, 2002) has identified and characterized puroindoline-like genes, cDNAs and/or polypeptides in a variety of other agriculturally important monocotyledonous crop species such as barley (Hordeum vulgare L.), oat (Avena sativa L.), rye (Secale cerealis L.). [We named the proteins in oats tryptophanin (Tanchak, 1998), also known as avenoindolines.] In contrast, other major monocotyledonous crops such corn (Zea mays L.), rice (Oryza sativa L.) and pasta wheat (Triticum durum L.) do not possess puroindoline-like sequences. [Recent evidence suggests that rice possesses a gene related to the PINs of wheat. It is believed that this rice sequence was not detected in earlier work as the degree of similarity is below the threshold of detection attainable with the hybridization conditions used in the earlier studies (Chantret, 2004).]
Other recent advances in our study of oat tryptophanins include the results of Triton X-114 phase-partitioning (Tanchak, 1998) and the immunolocalization of puroindoline-like proteins in oats seeds (Melnyk, manuscript in preparation). The phase-partitioning experiments show the presence of a major 14 kDa polypeptide in the detergent-rich phase. N-terminal sequencing of this polypeptide confirms that it is the mature polypeptide encoded by one of the oat tryptophanin cDNA clones isolated in our lab. Immunolocalization, using a monoclonal antibody preparation specific for "friabilin" (aka puroindoline), shows labeling on the surface of starch granules in the endosperm of oat seeds (MAb courtesy of Rhone Diagnostics, Glasgow). Although the exact specificity of this monoclonal preparation is not known (i.e. PIN-a and/or PIN-b), these results suggest that the relevant proteins in oats behave in a similar manner as the puroindolines in wheat. Because of the strong homology among the wild-type proteins from oat and wheat:
3 4 5 6 7 Altosaar 18022 Puroindoline b FPVTWPT-KWWKGGCEHEVREKCCKQLS-QIAPQCRCDSIRRVIQGRL
Puroindoline a FPVTWRWWKWWKGGC-QELLGECCSRLG-QMPPQCRCNIIQGSIQGDL
Tryptophanin 3B3 FPITWP-WKWWKGGCE-EVRNECCQLLG-QMPWECRCDAIWRSIQHEL
Tryptophanin 3B3T IPITWP-WKWWKGGCESEVRSQCCMELNIQIAPHCRCKAIWRAVQGEL
this suggests to us that we should explore their interchangeability in future work aimed at studying their 'cargo carrying capacity' - not their traditional fatty acid cargo, but fusion protein coding sequences.
PRODUCTION OF RECOMBINANT PROTEINS
Our studies on synthesis of storage proteins and their deposition in storage granules in seed endosperm tissue initially used high protein oat as a model because of DuPont's interest in such substitutes for soy protein. Because of our success in being able to isolate seed protein granules and transform plants with foreign DNA using Agrobacterium (Robert, 1989), we initiated a collaboration with Canadian Red Cross Blood Research Labs to produce high-value proteins like clotting factor VIII
(Altosaar, 1994). Having always included glutelin protein synthesis in rice as a comparator/model system (Robert, 1985) we developed recombinant DNA expression vectors based on the seed-specificity of the rice glutelin promoter. Supported by the Bayer (MILES
Pharmaceutical)/Red Cross R & D Fund, we were able in transgenic plants to produce cytokines like granulocyte macrophage colony stimulating factor, a recombinant protein used globally for bone marrow transplant patients (Sardana, 2002). Better than the 0.98 kilobase Gt3 glutelin promoter, the 1.8 kb Gtl promoter-driven plants contained human GM-CSF protein up to a level of 0.03% of total soluble protein. Because we have developed high efficiency transformation systems for producing transgenic rice plants (Cheng, 1998), the same promoters have been tested in rice seeds which then accumulated human GM-CSF
to a level of 1.3% of total soluble protein (unpublished results). Using the maize ubiquitin promoter instead in rice, we have achieved a foreign protein level of 3% of soluble proteins in the case of entomocide Bt (Cheng, 1998).
With funding from Roche's Genentech, Inc. we produced human insulin-like growth factors-I and -1B
using the same maize ubiquitin promoter vector (Panahi, 2003). The plant-made IGF-1 extracts were assayed for biological activity by Dr. Jenny Phipps' team at NRC's Steacie Institute for Molecular Sciences (now CEO of PharmaGap, Inc.) and the plant-made human IGF-l's were effective in stimulating the in vitro growth and proliferation of human SY5Y neuroblastoma cells. Remarkably this IGF- 1 B caused differentiation as well, implying a distinct biological role for pro-IGF-1B and suggesting it may play a role in tumorigenesis. As for recombinant hIGF-l, the highest expressing rice line contained 0.03% of total soluble protein, corresponding to 371 ng of rthIGF-1 (Panahi, 2004).
With our solid experience in seed protein synthesis, the production of recombinant PMP proteins in seeds was a logical extension of our existing research program. In addition, seed constituents play an important role in many medications. For example, seed-derived starch is used as an important component of excipient (non-drug) materials in drug delivery vehicles such as tablets, capsules and caplets. These considerations have led to an increased interest, by many research groups not just our own, in expressing desired gene sequences from diverse origins (e.g. human, bacteria, virus) in plants (Kusnadi, 1998) including rice seeds as a host platform (Yang, 2003). But to our knowledge no one to date has tried to use the starch granule surface of rice as a safe harbour or sub-organellar storage compartment for PMP accumulation and facilitating downstream purification.
Overall, knowledge of the sub-cellular location of recombinant proteins in transgenic plants is limited. However, a recent publication detailing the production of recombinant human lysozyme in rice seeds (Yang, 2003) is particularly noteworthy. Rice has two types of proteins bodies (PB). Type I
protein bodies are derived from the endoplasmic reticulum and contain prolamin storage proteins. Type II protein bodies are derived from the vacuolar membrane system and contain storage proteins of the globulin and glutelin classes. When human lysozyme was expressed with a signal peptide sequence, it accumulated in the Type II protein bodies. When the "strong" Gtl promoter was used, the resulting Altosaar 18022 higher level of lysozyme production appeared to distort storage-protein targeting/sorting resulting in changes in the distribution of native globulin and glutelin storage proteins and producing a change in the morphology of protein bodies. Therefore, higher levels of recombinant protein expression may have unexpected consequences. The overall level of protein ~d~ei ~~~" cqearaw site expression (controlled through promoter strength, etc.) should be a variable that is considered during the development of new N cargo protein protein expression technologies for use in seeds. These ~aflmO'o ~~-- disturbing results from Ventria Biosciences corroborates our own 15 year findings with the glutelin signal peptide PB
c targeting approach, the highest level we have achieved to date Surface is only 1.3% of total soluble protein (Sardana, PNAS
lipids Starch manuscript under revision). Thus a newer safer harbour for granule cargo proteins merits investigation and we believe puroindoline may guide us there, to the surface of Starch Granules (SG).
PREMISE FOR CURRENT RESEARCH PROPOSAL
Seeds are natural biomolecular storage organs, and can be stored for long periods of time. The storage proteins are segregated in specialized vacuoles called protein bodies.
Most seed storage proteins seem to have no enzymatic function, making the seed a promising target of recombinant proteins.
The starch granule surface is a dynamic protein compartment (Kotting, 2005).
The Ritte lab identified a variation of the in vitro binding capability of protein Rl to the starch granules of dark- and light-adapted leaves. Their research indicates that the starch granule surface may be used as a scaffold to organize transitory metabolic processes.
Starch granules form layers of amorphous and crystalline amylopectin matrix.
Amylose is not required for the development of the starch granule matrix but is present in a semi-helical and soluble form within the matrix. Amylose is synthesized starting in the core and continues outward through the amylopectin matrix as the granule grows. Amylose free starch granules contain amylopectin but filling of the matrix by amylose is slowed, yielding an amylose-free periphery.
However, the starch granule ultrastructure forms normally since the primary structural matrix contains only amlyopectin. Ji (2003) expressed a fusion protein of a starch granule binding protein domain and signal peptide which was able to gain entry into starch granules. It was shown that the fusion protein did not localize to the granule surface but did localize to the interior of the starch granule. Of note, higher concentrations of fusion protein were obtained in amylose free starch granules than in amylase-containing starch granules. The amyloplast, the amyloplast membrane, and the starch granule should be viewed as a storage compartment with valuable use for depositing and subsequently purifying pharmaceutical proteins in plants.
Recent research is revealing a complex starch granule surface. The discovery within starch granules of channels exposed to the protein matrix opens up new avenues for packing recombinant proteins targeted to amyloplasts (Huber and BeMiller, 2000). Given that our lab has been able to express functional pharmaceutical proteins in plant seeds, consideration should now be given to optimizing expression, extraction, and purification of these proteins.
In the previous attempts at expressing pharmaceutical proteins in our lab, the resulting product has most likely been targeted to the protein bodies of the seed, an obviously protein-rich compartment.
This result would complicate the process of extracting and purifying the relevant protein product as common protein solubilizations are aqueous based.
Now instead we would like to explore the possibility that if the pharmaceutical protein is expressed as a fusion protein with a puroindoline or oat tryptophanin as the "fusion partner", then this product might accumulate on the surface of starch granules in the seed - a relatively "protein-poor Altosaar 18022 compartment" (see Diag.). Isolation of the starch granule would then represent a significant enrichment event especially if air classification of dry flour streams is implemented as a pre-treatment of transgenic rice flour. If the junction of the two sequences in the fusion protein included a cleavable amino acid "linker" sequence then treatment of the starch granule preparation with the appropriate agent/protease could release the pharmaceutical polypeptide from the surface of the starch granule potentially in a "pure" or almost pure form. Our choice of puroindoline as the piggyback carrier or docking protein is also mitigated by the fact that Pin is the most abundant amphiphilic protein in wheat (Blochet, 1993).
Given that the wheat genome size is ca. 16.5 Gb, it is not surprising that its endosperm proteome displays over a thousand spots on two-dimensional electrophoretic gels (Islam, 2002), each in relatively low abundance. We then interpret this heightened molecular need for a very abundant phase-shuttle protein (Lipid Transfer Protein) as an indication of puroindoline's potential as a harbour-master to not only 'dock' lipids but perhaps as a novel protein targeting signal as well. We want to exploit puroindoline's abundance in soft wheat and oat to explore its ability to effect pharma-protein transfer to the Starch Granule Surface membrane lipid layer, using transgenic rice as the platform technology.
As rice (0. sativa) does not produce any puroindoline-like proteins, the starch granule in the rice endosperm is therefore likely to represent "new turf' for the planned PMP-fusion protein and may have a high binding capacity for this fusion protein. As we have previous experience producing transgenic rice, rice is the model system of choice (Cheng, 1998). Despite the caveat noted in "Starch Granule-associated proteins" section above concerning a PIN-like DNA sequence in the rice genome, our Western blots with anti-PIN Durotest monoclonal antibody were negative (unpublished results).
The selection of rice as a model system is not without its potential complications. In rice, the protein bodies do not disintegrate to form a protein matrix and, as noted above, there are, in fact, two distinct types of protein bodies in rice (e.g. Yang, 2003). Therefore, it is not clear how the fusion protein would gain access to the starch granule. However, with respect to the structural integrity of protein bodies, a similar situation exists in oats yet evidence from our immunolocalization work Immunofluorescent localization of PIN
homologues in oat (Left Panel).
2 m 'Hinoat' oat seed endosperm LR white section incubated in 1:500 dilution of Durotest Antibody and 1:100 fluorescein secondary Ab, 2sec exposure. Right: 0.1 s exposure phase contrast micrograph. Scale bar 20 m.
(Melnyk., manuscript in preparation) suggests that some sort of puroindoline-like protein surrounds the oat endosperm starch granules (note bright fluorescence encircles the 20 m diameter Starch Granules).
To see if PIN can localize to the surface of rice granules, we have conducted preliminary proof-of-concept in situ experiments with a PMP-PIN fusion protein. Letting recombinant Thioredoxin serve the role of a PMP candidate Cargo protein (see Diag. above), we constructed protein expression vectors coding for a His Tag-Thioredoxin-PIN-b fusion protein, and a Control vector expressing His Tag-Thx alone. PIN-b was subcloned from wheat genomic DNA into a pET-32b expression vector. This His Tag-Thioredoxin-PIN-b gene fusion construct was transformed into E. coli and the protein was expressed upon addition of IPTG inducer. The E.coli cell lysate, containing the Cargo-PIN was applied to T7 Promoter His-Tag Thioredoxin PinB T7 Terminator paraffin-embedded thin sections of rice kernels and allowed to incubate for 1 h at room temperature, followed by several 10 min washes to remove any unbound cell lysate products.
Upon fluorescent labeling with anti-His Tag primary and anti-mouse secondary FITC-conjugated antibodies, we observed consistent signal from the SG surface regions throughout the rice endosperm.
Applying His Tag-Thx Altosaar 18022 proteins alone yielded no significant signal. These observations support the hypothesis that Immunofluorescent localization of a recombinant PIN-b fusion to rice (Left Panel).
Rice paraffin-embedded endosperm 5 m section incubated in E. coli cell lysate containing Thx-PIN-b (Left Panel) or Thx only (Right Panel).
puroindolines have a docking domain with an affinity for rice starch granule surfaces. Such in situ experiments with sections are also underway in vitro with purified starch granules from wheat, oat, barley and rice, both non-transformed NT and transgenic, to explore the stringency or lack of specificity for PIN-SG interactions.
We have also started to study transgenic rice expressing puroindoline alone, from a wheat signal peptide-coding sequence construct. In rice kernels, PIN causes a softening of the rice seed as well as provides protection from fungal infection. This is consistent with the expected behavior for puroindoline, acting as a surfactant at the SG/protein matrix interface (see Diag. above). We have obtained, as a gift from Dr. Giroux, the last few kernels of this transgenic rice, line 97-1 (Krishnamurthy and Giroux, 2001). An immediate short-term objective will be to perform immunolocalization experiments to determine the sub-cellular location of PIN protein in the transgenic rice seed. We have been fortunate enough to have a 4'h year project student Charles Melnyk fix, embed, section and stain this valuable germplasm very meticulously. Upon fluorescent labeling with Durotest anti-PIN primary and anti-mouse secondary FITC-conjugated antibodies, we observed consistent signal from the SG
surface regions throughout the rice endosperm. These observations support the hypothesis that puroindolines have an affinity for this organelle. Combined with the new evidence emerging that the SG
surface may be 'spongier and more absorbent' than previously thought (Huber and BeMiller, 2000), it may be interesting to see how much PIN can be soaked up by rice SG surfaces in vitro and in vivo.
Immunofluorescent localization of Durotest anti-PIN antibody If transgenic immunolocalization experiments indicate that the puroindoline is not present on the surface of starch granules in significant abundance, then the research direction will be adjusted accordingly. Options include modifying the nature of the fusion construct (i.e. no signal sequence) so that translation occurs on cytosolic ribosomes rather than ribosomes bound to the surface of the endoplasmic reticulum. Under these circumstances the fusion protein, in theory, could associate with the surface of amyloplasts directly from the cytosol.
It will be interesting to discover where PIN-alone and PIN-fusions actually transport when we omit its signal peptide. In theory nascent PIN polypeptide carriers with or without their cargo-partner should readily be 'zippered onto' the lipid surface of the SG by virtue of the membrane-seeking tryptophan-box Docking domain (Diag.). This 'sans signal'option has obvious implications with respect to the formation of disulfide bonds - possibly altering the desired characteristics and properties of the fusion protein. Certainly "downstream processing" would be affected if the pharmaceutical protein normally possesses disulfide bonds necessitating restorative refolding procedures using glutathione/thioredoxin (Wu, 2005).
Altosaar 18022 In light of the above considerations, a variety of gene fusion constructs need to be generated to answer the many questions concerning translocation and retranslocation that may be operating in the rice endosperm cytoplasm/membrane compartments dynamics. PIN-a (-Trp-Arg-Trp-Trp-Lys-Trp-Trp-Lys), PIN-b (-Trp-Pro-Thr-Trp-Trp-Lys-) and oat tryptophanin (-Trp-Pro-Trp-Lys-Trp-Trp-Lys-) each have distinctive tryptophan-rich domains. It is believed that it is this domain that is primarily responsible for the lipid-binding properties of these proteins. Therefore, it would be appropriate to construct and test gene fusion constructs possessing each of these polypeptides to determine if there are any detectable differences in performance or behavior. My current NSERC Discovery grant pays for the MSc stipend of Mike Wall (BSc UCB) to construct a W>A PIN gene which will serve as a good control for transgenic rice, one where all the five tryptophan residues have been replaced by 5 Alanines using site-specific mutagenesis. In addition, the use of gene fusion constructs using promoters of different strength and/or tissue-specificity would also be desirable. Furthermore, a variety of constructs (e.g. N-terminal, C-terminal fusions, different pharmaceutical proteins) will be needed as, at this point, it is not known how the presence of the added sequence will affect the activity of the puroindoline/oat tryptophanin sequence. Retention of the ability to associate with starch granules is essential to the approach. Some cautionary flags we will watch out for include: The surprise results observed with our hCMV gB
targeting to Protein Bodies via cryptic signals (Wright, 2001) and those with the B subunit of E. codi heat labile enterotoxin (LT-B) expressed in maize endosperm put us on the alert to be ready for unexpected results (Chikwamba, 2003). In the latter case, the construct was engineered to retain the PMP in the Golgi but instead it ended up inside the starch granule as well as on its surface. Further research to elucidate mechanisms of protein translocation to the plastid, or retranslocation from a previous targeted locale to the SGS 'compartment' may produce strategically significant methodologies for PMP-enriched SGs.
Given our ten year's of experience with insulin-like growth factor-1 and -1B, and the continued collaborative support of Dr. Jenny Phipps and her team at PharmaGap, Inc. here in Ottawa, we expect to make serious headway in exploring this protein production and accumulation platform. We have already published two papers on plant-made IGF-ls and work well together. Furthermore, the good support from Genentech's IGF-1 team continues to be available.
PMP platforms based on field crops may disperse the human/industrial protein gene(s) via pollen, straw residues or through the food chain. So a variety of biosafety-related concerns (e.g.
confinement, containment, unintended exposure) will be uppermost in the various research designs.
Immediately we will set about to look at alternative hosts for the expression of the fusion proteins in starch-rich plants which are not edible or palatable. It may not be appropriate to produce bioactive compounds in a widely used agricultural crop such as rice, although here in our greenhouses our transgenic rice plants are completely isolated, over two thousand kms from the nearest rice fields (Stuttgart, AR). Despite this geographical isolation, we will immediately start searching for a parallel host system from the Poaceae (with 8,000 species) to look for one as closely related to rice as possible but universally considered to be unedible. We already have other key biosafety steps in place: our Bt genes in China can be confmed in hybrid rice male sterile lines (Zeng, 2002), and our HIV-family vaccine candidate protein gB has been contained in glasshouse and mineshafft (Tackaberry, 2003), The unique membrane-seeking properties of puroindoline's Trp-box, -WWWW- may facilitate a well-structured analysis of its synthesis, movement and localization, using the lipid surface of the rice starch granule as an easily attainable sub-organellar compartment. Rice flour milling, flour dust fractionation and water-washed starch granule procedures are centuries' old traditional technologies which are ripe for further study using the heterologous probe, puroindoline/tryptophanin. The major short-term and long-term objectives of this research program are summarized below.
Short-term objectives Altosaar 18022 (1) Immunolocalization experiments on transgenic rice samples to be replicated in detail to determine the sub-cellular location of the expressed puroindoline. Adjust nature of alternate plant host and design of gene fusion constructs if need be.
(2) Design and construction of "gene fusion" expression constructs encoding puroindoline or oat tryptophanin polypeptide sequence with a cleavable linker to "economically important polypeptides"
with no, one, two, or more disulfide bridges.
(3) Production of transgenic rice plants expressing constructs from (2).
Puroindoline a FPVTWRWWKWWKGGC-QELLGECCSRLG-QMPPQCRCNIIQGSIQGDL
Tryptophanin 3B3 FPITWP-WKWWKGGCE-EVRNECCQLLG-QMPWECRCDAIWRSIQHEL
Tryptophanin 3B3T IPITWP-WKWWKGGCESEVRSQCCMELNIQIAPHCRCKAIWRAVQGEL
this suggests to us that we should explore their interchangeability in future work aimed at studying their 'cargo carrying capacity' - not their traditional fatty acid cargo, but fusion protein coding sequences.
PRODUCTION OF RECOMBINANT PROTEINS
Our studies on synthesis of storage proteins and their deposition in storage granules in seed endosperm tissue initially used high protein oat as a model because of DuPont's interest in such substitutes for soy protein. Because of our success in being able to isolate seed protein granules and transform plants with foreign DNA using Agrobacterium (Robert, 1989), we initiated a collaboration with Canadian Red Cross Blood Research Labs to produce high-value proteins like clotting factor VIII
(Altosaar, 1994). Having always included glutelin protein synthesis in rice as a comparator/model system (Robert, 1985) we developed recombinant DNA expression vectors based on the seed-specificity of the rice glutelin promoter. Supported by the Bayer (MILES
Pharmaceutical)/Red Cross R & D Fund, we were able in transgenic plants to produce cytokines like granulocyte macrophage colony stimulating factor, a recombinant protein used globally for bone marrow transplant patients (Sardana, 2002). Better than the 0.98 kilobase Gt3 glutelin promoter, the 1.8 kb Gtl promoter-driven plants contained human GM-CSF protein up to a level of 0.03% of total soluble protein. Because we have developed high efficiency transformation systems for producing transgenic rice plants (Cheng, 1998), the same promoters have been tested in rice seeds which then accumulated human GM-CSF
to a level of 1.3% of total soluble protein (unpublished results). Using the maize ubiquitin promoter instead in rice, we have achieved a foreign protein level of 3% of soluble proteins in the case of entomocide Bt (Cheng, 1998).
With funding from Roche's Genentech, Inc. we produced human insulin-like growth factors-I and -1B
using the same maize ubiquitin promoter vector (Panahi, 2003). The plant-made IGF-1 extracts were assayed for biological activity by Dr. Jenny Phipps' team at NRC's Steacie Institute for Molecular Sciences (now CEO of PharmaGap, Inc.) and the plant-made human IGF-l's were effective in stimulating the in vitro growth and proliferation of human SY5Y neuroblastoma cells. Remarkably this IGF- 1 B caused differentiation as well, implying a distinct biological role for pro-IGF-1B and suggesting it may play a role in tumorigenesis. As for recombinant hIGF-l, the highest expressing rice line contained 0.03% of total soluble protein, corresponding to 371 ng of rthIGF-1 (Panahi, 2004).
With our solid experience in seed protein synthesis, the production of recombinant PMP proteins in seeds was a logical extension of our existing research program. In addition, seed constituents play an important role in many medications. For example, seed-derived starch is used as an important component of excipient (non-drug) materials in drug delivery vehicles such as tablets, capsules and caplets. These considerations have led to an increased interest, by many research groups not just our own, in expressing desired gene sequences from diverse origins (e.g. human, bacteria, virus) in plants (Kusnadi, 1998) including rice seeds as a host platform (Yang, 2003). But to our knowledge no one to date has tried to use the starch granule surface of rice as a safe harbour or sub-organellar storage compartment for PMP accumulation and facilitating downstream purification.
Overall, knowledge of the sub-cellular location of recombinant proteins in transgenic plants is limited. However, a recent publication detailing the production of recombinant human lysozyme in rice seeds (Yang, 2003) is particularly noteworthy. Rice has two types of proteins bodies (PB). Type I
protein bodies are derived from the endoplasmic reticulum and contain prolamin storage proteins. Type II protein bodies are derived from the vacuolar membrane system and contain storage proteins of the globulin and glutelin classes. When human lysozyme was expressed with a signal peptide sequence, it accumulated in the Type II protein bodies. When the "strong" Gtl promoter was used, the resulting Altosaar 18022 higher level of lysozyme production appeared to distort storage-protein targeting/sorting resulting in changes in the distribution of native globulin and glutelin storage proteins and producing a change in the morphology of protein bodies. Therefore, higher levels of recombinant protein expression may have unexpected consequences. The overall level of protein ~d~ei ~~~" cqearaw site expression (controlled through promoter strength, etc.) should be a variable that is considered during the development of new N cargo protein protein expression technologies for use in seeds. These ~aflmO'o ~~-- disturbing results from Ventria Biosciences corroborates our own 15 year findings with the glutelin signal peptide PB
c targeting approach, the highest level we have achieved to date Surface is only 1.3% of total soluble protein (Sardana, PNAS
lipids Starch manuscript under revision). Thus a newer safer harbour for granule cargo proteins merits investigation and we believe puroindoline may guide us there, to the surface of Starch Granules (SG).
PREMISE FOR CURRENT RESEARCH PROPOSAL
Seeds are natural biomolecular storage organs, and can be stored for long periods of time. The storage proteins are segregated in specialized vacuoles called protein bodies.
Most seed storage proteins seem to have no enzymatic function, making the seed a promising target of recombinant proteins.
The starch granule surface is a dynamic protein compartment (Kotting, 2005).
The Ritte lab identified a variation of the in vitro binding capability of protein Rl to the starch granules of dark- and light-adapted leaves. Their research indicates that the starch granule surface may be used as a scaffold to organize transitory metabolic processes.
Starch granules form layers of amorphous and crystalline amylopectin matrix.
Amylose is not required for the development of the starch granule matrix but is present in a semi-helical and soluble form within the matrix. Amylose is synthesized starting in the core and continues outward through the amylopectin matrix as the granule grows. Amylose free starch granules contain amylopectin but filling of the matrix by amylose is slowed, yielding an amylose-free periphery.
However, the starch granule ultrastructure forms normally since the primary structural matrix contains only amlyopectin. Ji (2003) expressed a fusion protein of a starch granule binding protein domain and signal peptide which was able to gain entry into starch granules. It was shown that the fusion protein did not localize to the granule surface but did localize to the interior of the starch granule. Of note, higher concentrations of fusion protein were obtained in amylose free starch granules than in amylase-containing starch granules. The amyloplast, the amyloplast membrane, and the starch granule should be viewed as a storage compartment with valuable use for depositing and subsequently purifying pharmaceutical proteins in plants.
Recent research is revealing a complex starch granule surface. The discovery within starch granules of channels exposed to the protein matrix opens up new avenues for packing recombinant proteins targeted to amyloplasts (Huber and BeMiller, 2000). Given that our lab has been able to express functional pharmaceutical proteins in plant seeds, consideration should now be given to optimizing expression, extraction, and purification of these proteins.
In the previous attempts at expressing pharmaceutical proteins in our lab, the resulting product has most likely been targeted to the protein bodies of the seed, an obviously protein-rich compartment.
This result would complicate the process of extracting and purifying the relevant protein product as common protein solubilizations are aqueous based.
Now instead we would like to explore the possibility that if the pharmaceutical protein is expressed as a fusion protein with a puroindoline or oat tryptophanin as the "fusion partner", then this product might accumulate on the surface of starch granules in the seed - a relatively "protein-poor Altosaar 18022 compartment" (see Diag.). Isolation of the starch granule would then represent a significant enrichment event especially if air classification of dry flour streams is implemented as a pre-treatment of transgenic rice flour. If the junction of the two sequences in the fusion protein included a cleavable amino acid "linker" sequence then treatment of the starch granule preparation with the appropriate agent/protease could release the pharmaceutical polypeptide from the surface of the starch granule potentially in a "pure" or almost pure form. Our choice of puroindoline as the piggyback carrier or docking protein is also mitigated by the fact that Pin is the most abundant amphiphilic protein in wheat (Blochet, 1993).
Given that the wheat genome size is ca. 16.5 Gb, it is not surprising that its endosperm proteome displays over a thousand spots on two-dimensional electrophoretic gels (Islam, 2002), each in relatively low abundance. We then interpret this heightened molecular need for a very abundant phase-shuttle protein (Lipid Transfer Protein) as an indication of puroindoline's potential as a harbour-master to not only 'dock' lipids but perhaps as a novel protein targeting signal as well. We want to exploit puroindoline's abundance in soft wheat and oat to explore its ability to effect pharma-protein transfer to the Starch Granule Surface membrane lipid layer, using transgenic rice as the platform technology.
As rice (0. sativa) does not produce any puroindoline-like proteins, the starch granule in the rice endosperm is therefore likely to represent "new turf' for the planned PMP-fusion protein and may have a high binding capacity for this fusion protein. As we have previous experience producing transgenic rice, rice is the model system of choice (Cheng, 1998). Despite the caveat noted in "Starch Granule-associated proteins" section above concerning a PIN-like DNA sequence in the rice genome, our Western blots with anti-PIN Durotest monoclonal antibody were negative (unpublished results).
The selection of rice as a model system is not without its potential complications. In rice, the protein bodies do not disintegrate to form a protein matrix and, as noted above, there are, in fact, two distinct types of protein bodies in rice (e.g. Yang, 2003). Therefore, it is not clear how the fusion protein would gain access to the starch granule. However, with respect to the structural integrity of protein bodies, a similar situation exists in oats yet evidence from our immunolocalization work Immunofluorescent localization of PIN
homologues in oat (Left Panel).
2 m 'Hinoat' oat seed endosperm LR white section incubated in 1:500 dilution of Durotest Antibody and 1:100 fluorescein secondary Ab, 2sec exposure. Right: 0.1 s exposure phase contrast micrograph. Scale bar 20 m.
(Melnyk., manuscript in preparation) suggests that some sort of puroindoline-like protein surrounds the oat endosperm starch granules (note bright fluorescence encircles the 20 m diameter Starch Granules).
To see if PIN can localize to the surface of rice granules, we have conducted preliminary proof-of-concept in situ experiments with a PMP-PIN fusion protein. Letting recombinant Thioredoxin serve the role of a PMP candidate Cargo protein (see Diag. above), we constructed protein expression vectors coding for a His Tag-Thioredoxin-PIN-b fusion protein, and a Control vector expressing His Tag-Thx alone. PIN-b was subcloned from wheat genomic DNA into a pET-32b expression vector. This His Tag-Thioredoxin-PIN-b gene fusion construct was transformed into E. coli and the protein was expressed upon addition of IPTG inducer. The E.coli cell lysate, containing the Cargo-PIN was applied to T7 Promoter His-Tag Thioredoxin PinB T7 Terminator paraffin-embedded thin sections of rice kernels and allowed to incubate for 1 h at room temperature, followed by several 10 min washes to remove any unbound cell lysate products.
Upon fluorescent labeling with anti-His Tag primary and anti-mouse secondary FITC-conjugated antibodies, we observed consistent signal from the SG surface regions throughout the rice endosperm.
Applying His Tag-Thx Altosaar 18022 proteins alone yielded no significant signal. These observations support the hypothesis that Immunofluorescent localization of a recombinant PIN-b fusion to rice (Left Panel).
Rice paraffin-embedded endosperm 5 m section incubated in E. coli cell lysate containing Thx-PIN-b (Left Panel) or Thx only (Right Panel).
puroindolines have a docking domain with an affinity for rice starch granule surfaces. Such in situ experiments with sections are also underway in vitro with purified starch granules from wheat, oat, barley and rice, both non-transformed NT and transgenic, to explore the stringency or lack of specificity for PIN-SG interactions.
We have also started to study transgenic rice expressing puroindoline alone, from a wheat signal peptide-coding sequence construct. In rice kernels, PIN causes a softening of the rice seed as well as provides protection from fungal infection. This is consistent with the expected behavior for puroindoline, acting as a surfactant at the SG/protein matrix interface (see Diag. above). We have obtained, as a gift from Dr. Giroux, the last few kernels of this transgenic rice, line 97-1 (Krishnamurthy and Giroux, 2001). An immediate short-term objective will be to perform immunolocalization experiments to determine the sub-cellular location of PIN protein in the transgenic rice seed. We have been fortunate enough to have a 4'h year project student Charles Melnyk fix, embed, section and stain this valuable germplasm very meticulously. Upon fluorescent labeling with Durotest anti-PIN primary and anti-mouse secondary FITC-conjugated antibodies, we observed consistent signal from the SG
surface regions throughout the rice endosperm. These observations support the hypothesis that puroindolines have an affinity for this organelle. Combined with the new evidence emerging that the SG
surface may be 'spongier and more absorbent' than previously thought (Huber and BeMiller, 2000), it may be interesting to see how much PIN can be soaked up by rice SG surfaces in vitro and in vivo.
Immunofluorescent localization of Durotest anti-PIN antibody If transgenic immunolocalization experiments indicate that the puroindoline is not present on the surface of starch granules in significant abundance, then the research direction will be adjusted accordingly. Options include modifying the nature of the fusion construct (i.e. no signal sequence) so that translation occurs on cytosolic ribosomes rather than ribosomes bound to the surface of the endoplasmic reticulum. Under these circumstances the fusion protein, in theory, could associate with the surface of amyloplasts directly from the cytosol.
It will be interesting to discover where PIN-alone and PIN-fusions actually transport when we omit its signal peptide. In theory nascent PIN polypeptide carriers with or without their cargo-partner should readily be 'zippered onto' the lipid surface of the SG by virtue of the membrane-seeking tryptophan-box Docking domain (Diag.). This 'sans signal'option has obvious implications with respect to the formation of disulfide bonds - possibly altering the desired characteristics and properties of the fusion protein. Certainly "downstream processing" would be affected if the pharmaceutical protein normally possesses disulfide bonds necessitating restorative refolding procedures using glutathione/thioredoxin (Wu, 2005).
Altosaar 18022 In light of the above considerations, a variety of gene fusion constructs need to be generated to answer the many questions concerning translocation and retranslocation that may be operating in the rice endosperm cytoplasm/membrane compartments dynamics. PIN-a (-Trp-Arg-Trp-Trp-Lys-Trp-Trp-Lys), PIN-b (-Trp-Pro-Thr-Trp-Trp-Lys-) and oat tryptophanin (-Trp-Pro-Trp-Lys-Trp-Trp-Lys-) each have distinctive tryptophan-rich domains. It is believed that it is this domain that is primarily responsible for the lipid-binding properties of these proteins. Therefore, it would be appropriate to construct and test gene fusion constructs possessing each of these polypeptides to determine if there are any detectable differences in performance or behavior. My current NSERC Discovery grant pays for the MSc stipend of Mike Wall (BSc UCB) to construct a W>A PIN gene which will serve as a good control for transgenic rice, one where all the five tryptophan residues have been replaced by 5 Alanines using site-specific mutagenesis. In addition, the use of gene fusion constructs using promoters of different strength and/or tissue-specificity would also be desirable. Furthermore, a variety of constructs (e.g. N-terminal, C-terminal fusions, different pharmaceutical proteins) will be needed as, at this point, it is not known how the presence of the added sequence will affect the activity of the puroindoline/oat tryptophanin sequence. Retention of the ability to associate with starch granules is essential to the approach. Some cautionary flags we will watch out for include: The surprise results observed with our hCMV gB
targeting to Protein Bodies via cryptic signals (Wright, 2001) and those with the B subunit of E. codi heat labile enterotoxin (LT-B) expressed in maize endosperm put us on the alert to be ready for unexpected results (Chikwamba, 2003). In the latter case, the construct was engineered to retain the PMP in the Golgi but instead it ended up inside the starch granule as well as on its surface. Further research to elucidate mechanisms of protein translocation to the plastid, or retranslocation from a previous targeted locale to the SGS 'compartment' may produce strategically significant methodologies for PMP-enriched SGs.
Given our ten year's of experience with insulin-like growth factor-1 and -1B, and the continued collaborative support of Dr. Jenny Phipps and her team at PharmaGap, Inc. here in Ottawa, we expect to make serious headway in exploring this protein production and accumulation platform. We have already published two papers on plant-made IGF-ls and work well together. Furthermore, the good support from Genentech's IGF-1 team continues to be available.
PMP platforms based on field crops may disperse the human/industrial protein gene(s) via pollen, straw residues or through the food chain. So a variety of biosafety-related concerns (e.g.
confinement, containment, unintended exposure) will be uppermost in the various research designs.
Immediately we will set about to look at alternative hosts for the expression of the fusion proteins in starch-rich plants which are not edible or palatable. It may not be appropriate to produce bioactive compounds in a widely used agricultural crop such as rice, although here in our greenhouses our transgenic rice plants are completely isolated, over two thousand kms from the nearest rice fields (Stuttgart, AR). Despite this geographical isolation, we will immediately start searching for a parallel host system from the Poaceae (with 8,000 species) to look for one as closely related to rice as possible but universally considered to be unedible. We already have other key biosafety steps in place: our Bt genes in China can be confmed in hybrid rice male sterile lines (Zeng, 2002), and our HIV-family vaccine candidate protein gB has been contained in glasshouse and mineshafft (Tackaberry, 2003), The unique membrane-seeking properties of puroindoline's Trp-box, -WWWW- may facilitate a well-structured analysis of its synthesis, movement and localization, using the lipid surface of the rice starch granule as an easily attainable sub-organellar compartment. Rice flour milling, flour dust fractionation and water-washed starch granule procedures are centuries' old traditional technologies which are ripe for further study using the heterologous probe, puroindoline/tryptophanin. The major short-term and long-term objectives of this research program are summarized below.
Short-term objectives Altosaar 18022 (1) Immunolocalization experiments on transgenic rice samples to be replicated in detail to determine the sub-cellular location of the expressed puroindoline. Adjust nature of alternate plant host and design of gene fusion constructs if need be.
(2) Design and construction of "gene fusion" expression constructs encoding puroindoline or oat tryptophanin polypeptide sequence with a cleavable linker to "economically important polypeptides"
with no, one, two, or more disulfide bridges.
(3) Production of transgenic rice plants expressing constructs from (2).
(4) Evaluation of transgenic rice at the level of DNA, RNA, and protein to confirm presence and function of gene constructs.
(5) Confirmation of tissue-specificity of expression, subcellular immunolocalization, bioassays.
(6) Purification of seed starch granules - confirmation of presence of "fusion protein", granule affinity.
(7) Development of protocol for purification of "economically important polypeptide" from granules using dry-milling principles from rice flour craftsmen in China, California and Arkansas.
(8) Construction, and use of "second generation" or "optimized" gene constructs as (ifj needed.
(9) Bioassays and protein purity, stability, storage studies for pharmaceutical grade quality.
Long-term objectives (1) Confirmation of "structural authenticity" and biological/therapeutic activity of "economically important polypeptide" e.g IGF-1, GM-CSF, human milk sCD14, lysozyme, leptin, grehlin.
(2) Pursue Patent applications as aggressively as possible.
(3) Consideration of alternative "host" systems from members of Poaceae family.
(4) Consideration of potential problems to "scaling up" the process.
Methods and proposed approach Protein characterization methods: Our labs are equipped with FPLC, HPLC, SDS-PAGE and electrospray MS. We share labs and a PhD student, Pham Van Thong, with Prof.
Harvey Kaplan, a renowned protein chemist, and his expertise is also available. In the past we have raised rabbit antibodies to oat globulins, prolamins and tryptophanin so we do not anticipate problems with the immunocytochemistry work.
Plant material: The oat-breeding program just next to us at the Central Experimental Farm in Ottawa will continue to supply us with oat and wheat lines that are hard and soft in endosperm texture (L Reid, V Burrows). The spring wheat germplasm, originally developed by Dr. Dubuc in Ste-Foy, is now studied here also by the wheat breeders in Ottawa (H. Voldeng, R. Pandeya).
Flour milling labs are available at the Eastern Wheat Quality Lab on The CE Farm (Don Flynn, L.
Pietrzack, AA-F Canada).
Rice breeding and genetics support is integral to our research program. We are privileged to have the support of Karen Moldenhauer at Arkansas State Rice Research and Extension Center (Stuttgart) who supplies us constantly with rice seed varieties. Professor Qingyao Shu's rice breeding program and transgenic rice field trial plots at Zhejiang University in Hangzhou, China are an exceptional benefit of having been in the Rockefeller Foundation International Rice Biotech Network since 1991.
Transgenics: Simply because rice transformation with Agrobacterium is routine in our it does not mean it will be easy to transform durum wheat (if needed in this workplan). Many labs have reported success with biolistics so we sent our NSERC summer student, Melissa Toupin to the latter lab in 2003 and Prof.
Sautter's wheat regeneration system has been transferred to Ottawa. One of the more popular methods, from Monsanto labs, uses Agrobacterium (Hu, 2003) and shares many similarities with our rice methods (Cheng, 1998). Dr. Hu and her co-author Sally G. Metz at Monsanto now fund our lab on wheat seed globulin genoniics and proteomics (in relation to Juvenile Diabetes associated protein insults to the human immune system) so we have direct access to their in-house expertise on wheat transformation Altosaar 18022 and glyphosate selection. In all of our different approaches, we expect that any success in creating stable transformants will depend greatly on screening of susceptible durum genotypes and precise handling of the calli during co-cultivation.
Molecular strategies:
We use routinely the maize ubiquitin promoter (courtesy P. Quail, USDA) and the rice glutelin promoters gt3 and gtl (courtesy T. Okita, WSU) to drive high expression of foreign proteins in transgenic endosperm. The Ubi promoter has given us as much as 3% of total soluble protein when expressing a bacterial protein in rice endosperm (Cheng, 1998). So with transgenic hard durum wheat we expect that any level of PIN protein approaching this concentration should manifest itself on the Perten hardness SKCS values for which we have the instrumentation in lab. In our ongoing work expressing human insulin-like growth factor-1 protein in endosperm, the Ubi promoter has yielded the recombinant protein to a level up to 0.024% of extracted tobacco leaf protein (Panahi, 2003). The Ubi-IGF-1 cassettes were also put into transgenic rice plants and endosperm data there will be useful for comparisons among glutelin Gtl/Gt3/ and Ubi-PIN driven protein values.
For routine subcloning and expression in E. coli we use plasmid pKK233-2 as well as the more standard pET32 vectors (K. Wu, D. Blais, M. Tanchak are our experts). For routine rice transformations our recombinant expression constructs for IGF-1, GM-CSF, sCD14 human milk coding sequences are subcloned into independent pCAMBIA 1303 monocot transformation vectors.
Constructs are incorporated into Agrobacterium tumefaciens strain LBA 4404 by the freeze-thaw method from Clontech.
Protein purification: Puroindolines, being related to membrane-altering plant-derived anti-fungal peptides in plants, have a powerful affinity for membranes. Triton X-114 phase partitioning was originally used by Blochet (1993) to extract these low molecular weight proteins from endosperm flour of bread wheat (Triticum aestivum). The detergent phase, containing membrane-associated proteins, was predominantly puroindolines. So we anticipate in rice flour, after air cyclone classification of the densest SGs, the Cargo-Docking fusion proteins will be enriched and relatively pure requiring little polishing, but FastProtein chromatography and HPLC will follow standard protein chemistry protocols.
Anticipated Yields and Process Economics:
In the transgenic LT-B maize line, 1.3 and 2 g of LT-B was obtained from 1 kg of starch and endosperm, respectively (Chikwamba, 2003). Because most aqueous soluble proteins are lost in the starch purification process (in this case water-washed starch as in wet milling), the large amount of soluble LT-B copurified with starch fraction suggested a tight association of LT-B with starch.
Competitive Intellectual Property considerations do arise with such related findings, so there is a possibility that this LT-B/maize-SG interaction may be in process of being patented. However, given that the Mason-Arntzen group in Arizona is dedicated to oral delivery of global vaccines, it is unlikely that they will develop a PMP platform of it. In our current case with rice, this PMP-PIN/rice-SG
separation process (PowerFlour) has been disclosed to the University of Ottawa's Tech Transfer Office and is covered by an Invention Disclosure.
NATURE OF GROUP COLLABORATION
Pertinent expertise of team members The Altosaar lab brings a comprehensive skill set to this Strategic project on Novel BioProducts and BioProcessing. We have expertise in the purification of seed proteins, SDS-PAGE, production of antisera, immunoblotting, and recombinant DNA technology including transgenic gene expression. This past year we have become experienced with the immunofluorescent localization of proteins in seed cells (Melnyk., manuscript in preparation). Altosaar has been studying oat, wheat and rice since 1978, with more than 55 publications alone covering just the biology, microscopy and genetics of seed proteins.
Altosaar 18022 The lab has extensive experience with the production of transgenic plants including the production of recombinant human proteins in seeds of rice and tobacco.
Collaboration between team members:
Dr. Phipps also has a strong research track record here in the Ottawa area.
Originally based at NRC on Sussex Drive, she was instrumental in creating PharmaGap, Inc. to commercialize her team's capabilities in molecular biology, biopharmaceuticals and biotechnology.
Therefore, there will be considerable opportunity for on-site collaboration and co-ordination of our research efforts at low cost to the NSERC grant, e.g. no expenses for accommodations, meals, etc. Dr. Phipps has been a long-time adjunct member of our Biochemistry Departrnent. In general, the Altosaar lab will design and perform construction of gene constructs required for the production of transgenic plants, and will characterize transgenic plants (e.g. immunoblotting, nucleic acid blotting, PCR, Westerns, flour fractionation, protein purification). Dr. Phipps has assisted with bioassays to ensure the purified PMP proteins are active and not denatured by the various purification protocols to be applied.
Training: Altosaar has an extensive history of training undergraduate, postgraduate and postdoctoral students and is requesting funding for one undergraduate summer research assistant, one postgraduate student and one postdoctoral fellow for the duration of the grant. [Please refer to the accompanying Budget Justification section for details.] Our previous NSERC Group Research Grant in Ottawa has supported the work of one PhD student, David Blais, and the salary of one MSc student, Kechun Wu.
Although David had his own NSERC postgrad scholarship, his research lab costs were substantial because not only did he produce the antibacterial protein from mother's milk, sCD 14, in transgenic plants, but has expanded our knowledge of LPS-receptor to discover the molecular basis of the 'immune-privileged' nature of the human cornea. Mr. Wu cloned the PIN-b gene from a model diploid wheat, T. monococcum, and has used various pET expression vectors and many refolding cocktails to get enough soluble PIN-b ready for stable isotope labeling. We are working with Professor Natalie Goto's NMR lab in our Chemistry Department to achieve the first three-dimensional solution structure of PIN which will greatly assist our interpretation of PIN transport and its interphase phenomena in transgenic rice flour. The three new students to be hired on this Strategic project will also receive a world-class competitive training in applied biotechnology.
Long-term objectives (1) Confirmation of "structural authenticity" and biological/therapeutic activity of "economically important polypeptide" e.g IGF-1, GM-CSF, human milk sCD14, lysozyme, leptin, grehlin.
(2) Pursue Patent applications as aggressively as possible.
(3) Consideration of alternative "host" systems from members of Poaceae family.
(4) Consideration of potential problems to "scaling up" the process.
Methods and proposed approach Protein characterization methods: Our labs are equipped with FPLC, HPLC, SDS-PAGE and electrospray MS. We share labs and a PhD student, Pham Van Thong, with Prof.
Harvey Kaplan, a renowned protein chemist, and his expertise is also available. In the past we have raised rabbit antibodies to oat globulins, prolamins and tryptophanin so we do not anticipate problems with the immunocytochemistry work.
Plant material: The oat-breeding program just next to us at the Central Experimental Farm in Ottawa will continue to supply us with oat and wheat lines that are hard and soft in endosperm texture (L Reid, V Burrows). The spring wheat germplasm, originally developed by Dr. Dubuc in Ste-Foy, is now studied here also by the wheat breeders in Ottawa (H. Voldeng, R. Pandeya).
Flour milling labs are available at the Eastern Wheat Quality Lab on The CE Farm (Don Flynn, L.
Pietrzack, AA-F Canada).
Rice breeding and genetics support is integral to our research program. We are privileged to have the support of Karen Moldenhauer at Arkansas State Rice Research and Extension Center (Stuttgart) who supplies us constantly with rice seed varieties. Professor Qingyao Shu's rice breeding program and transgenic rice field trial plots at Zhejiang University in Hangzhou, China are an exceptional benefit of having been in the Rockefeller Foundation International Rice Biotech Network since 1991.
Transgenics: Simply because rice transformation with Agrobacterium is routine in our it does not mean it will be easy to transform durum wheat (if needed in this workplan). Many labs have reported success with biolistics so we sent our NSERC summer student, Melissa Toupin to the latter lab in 2003 and Prof.
Sautter's wheat regeneration system has been transferred to Ottawa. One of the more popular methods, from Monsanto labs, uses Agrobacterium (Hu, 2003) and shares many similarities with our rice methods (Cheng, 1998). Dr. Hu and her co-author Sally G. Metz at Monsanto now fund our lab on wheat seed globulin genoniics and proteomics (in relation to Juvenile Diabetes associated protein insults to the human immune system) so we have direct access to their in-house expertise on wheat transformation Altosaar 18022 and glyphosate selection. In all of our different approaches, we expect that any success in creating stable transformants will depend greatly on screening of susceptible durum genotypes and precise handling of the calli during co-cultivation.
Molecular strategies:
We use routinely the maize ubiquitin promoter (courtesy P. Quail, USDA) and the rice glutelin promoters gt3 and gtl (courtesy T. Okita, WSU) to drive high expression of foreign proteins in transgenic endosperm. The Ubi promoter has given us as much as 3% of total soluble protein when expressing a bacterial protein in rice endosperm (Cheng, 1998). So with transgenic hard durum wheat we expect that any level of PIN protein approaching this concentration should manifest itself on the Perten hardness SKCS values for which we have the instrumentation in lab. In our ongoing work expressing human insulin-like growth factor-1 protein in endosperm, the Ubi promoter has yielded the recombinant protein to a level up to 0.024% of extracted tobacco leaf protein (Panahi, 2003). The Ubi-IGF-1 cassettes were also put into transgenic rice plants and endosperm data there will be useful for comparisons among glutelin Gtl/Gt3/ and Ubi-PIN driven protein values.
For routine subcloning and expression in E. coli we use plasmid pKK233-2 as well as the more standard pET32 vectors (K. Wu, D. Blais, M. Tanchak are our experts). For routine rice transformations our recombinant expression constructs for IGF-1, GM-CSF, sCD14 human milk coding sequences are subcloned into independent pCAMBIA 1303 monocot transformation vectors.
Constructs are incorporated into Agrobacterium tumefaciens strain LBA 4404 by the freeze-thaw method from Clontech.
Protein purification: Puroindolines, being related to membrane-altering plant-derived anti-fungal peptides in plants, have a powerful affinity for membranes. Triton X-114 phase partitioning was originally used by Blochet (1993) to extract these low molecular weight proteins from endosperm flour of bread wheat (Triticum aestivum). The detergent phase, containing membrane-associated proteins, was predominantly puroindolines. So we anticipate in rice flour, after air cyclone classification of the densest SGs, the Cargo-Docking fusion proteins will be enriched and relatively pure requiring little polishing, but FastProtein chromatography and HPLC will follow standard protein chemistry protocols.
Anticipated Yields and Process Economics:
In the transgenic LT-B maize line, 1.3 and 2 g of LT-B was obtained from 1 kg of starch and endosperm, respectively (Chikwamba, 2003). Because most aqueous soluble proteins are lost in the starch purification process (in this case water-washed starch as in wet milling), the large amount of soluble LT-B copurified with starch fraction suggested a tight association of LT-B with starch.
Competitive Intellectual Property considerations do arise with such related findings, so there is a possibility that this LT-B/maize-SG interaction may be in process of being patented. However, given that the Mason-Arntzen group in Arizona is dedicated to oral delivery of global vaccines, it is unlikely that they will develop a PMP platform of it. In our current case with rice, this PMP-PIN/rice-SG
separation process (PowerFlour) has been disclosed to the University of Ottawa's Tech Transfer Office and is covered by an Invention Disclosure.
NATURE OF GROUP COLLABORATION
Pertinent expertise of team members The Altosaar lab brings a comprehensive skill set to this Strategic project on Novel BioProducts and BioProcessing. We have expertise in the purification of seed proteins, SDS-PAGE, production of antisera, immunoblotting, and recombinant DNA technology including transgenic gene expression. This past year we have become experienced with the immunofluorescent localization of proteins in seed cells (Melnyk., manuscript in preparation). Altosaar has been studying oat, wheat and rice since 1978, with more than 55 publications alone covering just the biology, microscopy and genetics of seed proteins.
Altosaar 18022 The lab has extensive experience with the production of transgenic plants including the production of recombinant human proteins in seeds of rice and tobacco.
Collaboration between team members:
Dr. Phipps also has a strong research track record here in the Ottawa area.
Originally based at NRC on Sussex Drive, she was instrumental in creating PharmaGap, Inc. to commercialize her team's capabilities in molecular biology, biopharmaceuticals and biotechnology.
Therefore, there will be considerable opportunity for on-site collaboration and co-ordination of our research efforts at low cost to the NSERC grant, e.g. no expenses for accommodations, meals, etc. Dr. Phipps has been a long-time adjunct member of our Biochemistry Departrnent. In general, the Altosaar lab will design and perform construction of gene constructs required for the production of transgenic plants, and will characterize transgenic plants (e.g. immunoblotting, nucleic acid blotting, PCR, Westerns, flour fractionation, protein purification). Dr. Phipps has assisted with bioassays to ensure the purified PMP proteins are active and not denatured by the various purification protocols to be applied.
Training: Altosaar has an extensive history of training undergraduate, postgraduate and postdoctoral students and is requesting funding for one undergraduate summer research assistant, one postgraduate student and one postdoctoral fellow for the duration of the grant. [Please refer to the accompanying Budget Justification section for details.] Our previous NSERC Group Research Grant in Ottawa has supported the work of one PhD student, David Blais, and the salary of one MSc student, Kechun Wu.
Although David had his own NSERC postgrad scholarship, his research lab costs were substantial because not only did he produce the antibacterial protein from mother's milk, sCD 14, in transgenic plants, but has expanded our knowledge of LPS-receptor to discover the molecular basis of the 'immune-privileged' nature of the human cornea. Mr. Wu cloned the PIN-b gene from a model diploid wheat, T. monococcum, and has used various pET expression vectors and many refolding cocktails to get enough soluble PIN-b ready for stable isotope labeling. We are working with Professor Natalie Goto's NMR lab in our Chemistry Department to achieve the first three-dimensional solution structure of PIN which will greatly assist our interpretation of PIN transport and its interphase phenomena in transgenic rice flour. The three new students to be hired on this Strategic project will also receive a world-class competitive training in applied biotechnology.
Claims (17)
1. A fusion polypeptide comprising:
(a) a lipid-binding tryptophan rich domain;
(b) a cargo polypeptide fused to said tryptophan rich domain.
(a) a lipid-binding tryptophan rich domain;
(b) a cargo polypeptide fused to said tryptophan rich domain.
2. The fusion polypeptide of claim 1 wherein said cargo polypeptide is a biologically active, commercially significant, or industrially useful polypeptide.
3. The fusion polypeptide of claim 2 wherein said cargo polypeptide is selected from the group consisting of a cosmeceutical, an antibody, a vaccine, a protein pharmaceutical, and a recombinant bioproduct.
4. The fusion polypeptide of claim 1 wherein said tryptophan rich domain is selected from the group consisting of puroindoline-a, puroindoline-b, avenoindoline-a, avenoindoline-b, tryptophanin, hordoindoline, or grain softness protein.
5. The fusion polypeptide of claim 1 comprising a cleavage site between said tryptophan rich domain and said cargo polypeptide.
6. A fusion polypeptide comprising a tryptophan rich domain of a starch granule surface lipid-associated polypeptide, wherein said tryptophan rich domain is fused to a polypeptide and said polypeptide is not endogenous to starch granules.
7. The fusion polypeptide of claim 6 further comprising a starch granule bound to said polypeptide.
8. The fusion polypeptide of claim 6 wherein said tryptophan rich domain is said starch granule surface lipid-associated polypeptide.
9. The fusion polypeptide of claim 6 further comprising a cleavage site between said tryptophan rich domain and said polypeptide.
10. The fusion polypeptide of claim 6 wherein said starch granule surface lipid-associated polypeptide is selected from the group consisting of puroindoline-a, puroindoline-b, avenoindoline-a, avenoindoline-b, tryptophanin, hordoindoline, or grain softness protein.
11. The fusion polypeptide of claim 8 wherein said starch granule surface lipid-associated polypeptide is selected from the group consisting of puroindoline-a, puroindoline-b, avenoindoline-a, avenoindoline-b, tryptophanin, hordoindoline, or grain softness protein.
12. The fusion polypeptide polypeptide of claim 6 wherein said starch granule surface lipid-associated polypeptide is selected from the group consisting of puroindoline-a, puroindoline-b, avenoindoline-a, avenoindoline-b, tryptophanin, hordoindoline, or grain softness protein.
13. The fusion polypeptide of claim 6 wherein said polypeptide is selected from the group consisting of a cosmeceutical, an antibody, a vaccine, a protein pharmaceutical, and a recombinant bioproduct.
14. The hybrid polypeptide of claim 7 wherein said polypeptide is selected from the group consisting of a cosmeceutical, an antibody, a vaccine, a protein pharmaceutical, and a recombinant bioproduct.
15. The hybrid polypeptide of claim 8 wherein said polypeptide is selected from the group consisting of a cosmeceutical, an antibody, a vaccine, a protein pharmaceutical, and a recombinant bioproduct.
16. The hybrid polypeptide of claim 9 wherein said polypeptide is selected from the group consisting of a cosmeceutical, an antibody, a vaccine, a protein pharmaceutical, and a recombinant bioproduct.
17. The fusion polypeptide of claim 1 wherein said tryptophan rich domain is said starch granule surface lipid-associated polypeptide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2555137 CA2555137A1 (en) | 2006-08-07 | 2006-08-07 | Puroindolines as fusion-protein carriers in molecular pharming |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2555137 CA2555137A1 (en) | 2006-08-07 | 2006-08-07 | Puroindolines as fusion-protein carriers in molecular pharming |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2555137A1 true CA2555137A1 (en) | 2008-02-07 |
Family
ID=39030906
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2555137 Abandoned CA2555137A1 (en) | 2006-08-07 | 2006-08-07 | Puroindolines as fusion-protein carriers in molecular pharming |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2555137A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150099860A1 (en) * | 2013-10-08 | 2015-04-09 | Proteins Easy Corp | Methods for separating and purifying endogenous, exogenous and recombinant proteins/peptides from plants and animals using aqueous-free, anhydrous strategies |
-
2006
- 2006-08-07 CA CA 2555137 patent/CA2555137A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150099860A1 (en) * | 2013-10-08 | 2015-04-09 | Proteins Easy Corp | Methods for separating and purifying endogenous, exogenous and recombinant proteins/peptides from plants and animals using aqueous-free, anhydrous strategies |
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