Room: RGN 4232 (office) 4251 (lab)
Office: 613-562-5800 ext. 6371 (office), 6375 (lab)
Personal E-mail: email@example.com
Molecular Bio-Pharming and Protein Farming
Production of recombinant proteins in transgenic plants: Proteins manufactured by Plant Molecular Farming are not only free of viral contamination but also enjoy improved recovery economics. cDNA's coding for commercially-important polypeptides are cloned into suitable plant transformation vectors. Our lab is developing cheap, safe and large-scale biological production of Plant-Made Proteins in transgenic seed compartments. Proteins-of-Interest (POI) include, biocatalysers, growth factors, antimicrobials and vaccine antigens. Tryptophan rich peptide domains target the POI onto deposition sites (starch granule) in the endosperm cell environment. Goals include maximal synthesis of POI in the seed and its maximal recovery from flour, using unique anhydrous cleavage.
- Altosaar I, Loit E, Greenham TJ, Hansen AE. Anhydrous strategies for separating purifying and commercializing recombinant proteins – New frontiers in aqueous-free extraction. Plant Based Vaccines Antibodies Biologics Meeting, University of Latvia, Riga, 10-12 June 2019.
- Greenham TJ, Altosaar I. 2018. Wheat puroindolines tether to starch granule surfaces in puroindoline-null (Pin-null) plants. J. Cereal Science 79: 286-293 doi.org/10.1016/j.jcs.2017.06.001
- Wan S, Truong-Trieu V, Ward TL, Whalen JK, Altosaar I. 2017. Advances in the use of genetically modified biomass for biodiesel generation. Biofuels Bioprod Bioref. 11:749–764 invited review. DOI: 10.1002/bbb.1777
- US 2016/0251396 A1 Methods for Separating and Purifying Endogenous, Exogenous and Recombinant Proteins/peptides from Plants and Animals using Aqueous-free, Anhydrous Strategies, Published: Sep 1, 2016, Inventors I Altosaar, T Greenham. PCT application.
- Boukebbous K, Moresco JJ, Yates JR III, Altosaar I. Protein extraction efficiency of soft and hard seeds using the Precellys lysing kits for MudPIT proteomics (388 KB). 2014. Bertin Technol Appl Note 03712-810-DU084_Protein_Seeds_MedHealth_US
- Greenham T, Altosaar I. 2013. Molecular strategies to engineer transgenic rice seed compartments for large-scale production of plant-made pharmaceuticals (251 KB). Methods in Molec Biol 956: 311-326. DOI: 10.1007/978-1-62703-194-3_22
The Altosaar Lab has been "stuck on starch" for some time. We've been studying the surface of the starch granule since 1984. The "protein community network" and the proteomic "Systems" landscape at the solid granule surface is becoming a hot spot for bioenergy research. We view the amyloplast as a bio-battery, solid fuel cells storing solar energy as fixed carbon.
Starch Granule Associated Proteins (SGAPs) were first reported by our lab in 1984. LCA-treated GMA sections of starch granules from wheat showed growth rings (arrowhead) in the large type A starch granules. Lectin binding also occurs in the smaller type B granules (arrow) but no growth rings are visible. "Halos" are discernible around both types of granules (Fig. 7) and we have been in hot pursuit of this proteomic-calyx ever since, using advanced biochemical and molecular genetic strategies.
These are not "random" proteins stuck to the surface of the bio-battery. When expressed in transgenic corn (Fig. F), they can have dramatic regulatory effects that need to be urgently dissected. From wheat the starch granule associated protein sporting a unique tryptophan-rich binding domain, puroindoline (Pin), regulates a 25% increase in oil! – implications to biomass and biodiesel outcomes are obvious.
Figure 5- WGA-treated cryostat section of Candle rapeseed, counterstained with Fast Green, showing fluorescent lectin binding to the seed coat mucilage (arrow). No specific fluorescence is visible in the cotyledon (C). 6- LCA-treated section of oat showing lectin binding to starch granules (S). No fluorescence is visible in the embryo (E) or in the endosperm matrix surrounding each compound starch granule. 7- LCA-treated GMA section of starch granules from wheat showing growth rings (arrowhead) in the large type A granules. Lectin binding also occurs in the smaller type B granules (arrow) but no growth rings are visible. “Halos” are discernible around both types of granules. 8- WGA binding to fungal hyphae penetrating the pericarp (arrow) and packed between the cuticular layers of the testa (arrowhead) of wheat. Fluorescence in the aleurone cells (A) is autofluorescence, and is not due to lectin binding. 9- GMA section of sprouted wheat showing the fungal hyphae (arrow), after WGA treatment, penetrating the non-fluorescent starch granule (arrowhead). Autofluorescence is visible in the aleurone layer (A). 10- WGA binding to fungal hyphae (arrows) between starch granules (S) in the starchy endosperm of wheat. Individual hyphae are readily distinguishable.
- Miller Yiu Fulcher Altosaar 1984 Food Microstructure 3:133-9 (Figs. 5-10 panel)
- Wall ML, Wheeler HL, Smith JC, Figeys D, Altosaar I. 2010. Mass spectrometric analysis reveals remnants of host-pathogen molecular interactions at the starch granule surface in wheat endosperm. Phytopathology 100: 848-854
- Wall ML, Wheeler H, Huebsch MP, Smith JC, Figeys D. Altosaar I. 2010. The tryptophan rich domain of puroindoline is directly associated with the starch granule surface as judged by tryptic shaving and mass spectrometry. Journal of Cereal Science 52: 115-120
"The starch grain [...] opens the door to the establishment of a new discipline, [...] the molecular mechanics of organized bodies." Carl Nägeli, 1858.
Microbiota of soil rhizospheres
Clearly the potential impacts on human health from climate change are of significant concern. The top three Green House Gases are carbon dioxide, methane and nitrous oxide (N2O). Although very little N2O is released to the atmosphere, it is the most potent GHG and accounts for approximately 7% of yearly GHG potentials. It has 310 times the warming potential of CO2 per molecule and an atmospheric half-life of 120 years. Besides its heat absorbing capacity, N2O helps deplete stratospheric ozone. Atmospheric concentrations of N2O have grown by 17% since 1750 and at present, N2O increases 0.3% per year. Food production contributes a small proportion of GHGs but is responsible for 80% of N2O emissions, primarily through use of nitrogen fertilizers. Dissimilative denitrification is the reduction of nitrate (NO3-) to dinitrogen gas (N2) through the anaerobic respiration of N. We are amplifying N2O breakdown, engineered masterfully by microbes in soil such as Pseudomonas over eons of evolution, by transferring the nos gene into crop plants using rhizospheres signal peptides to amass a stable soil ameliorant into food production cycles. Call it Atmospheric Medicine via Genetically Engineered soils.
- Demone JJ, Wan S, Nourimand M, Hansen AE, Shu QY, Altosaar I. 2018. New breeding techniques for greenhouse gas (GHG) mitigation: plants may express nitrous oxide reductase. Climate 6(4): 80; https://doi.org/10.3390/cli6040080 17pp
- Wan S, Greenham T, Goto K, Mottiar Y, Johnson AM, Staebler JM, Zaidi MA, Shu QY, Altosaar I. 2014. A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase from Pseudomonas stutzeri in transgenic plants. Can J Plant Sci, 94(6): 1013-1023, 10.4141/cjps2013-141
- Wan S, Ward TL, Altosaar I 2012 Strategy and tactics of disarming Greenhouse Gases (GHG) at the source: N2O reductase-crops. Trends in Biotechnology 30: 410-415
- Wan S et al 2012 Phytoremediation of nitrous oxide: expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase. Transgenic Research 21:593–603
- Wan S et al 2012 Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes. Ecology & Evolution 2:286–297
- Wan S et al 2012 Bacterial nitrous oxide reductase expressed in transgenic plants: evidence for sufficient anaerobicity to permit activity. Can J Plant Sci 92: 1283-1294.
Innate immunity of mammary and gastrointestinal epithelia
Nutritional and immunological modulation of neonatal gut health intrigues us. We endeavour to shed more light on the "black box" between breast milk and baby's first bacterial invaders. Several basic and translational projects focus on how mother's milk and/or infant formula and enteric microflora interact with gastric associated lymphoid tissue to impart gut health. Molecular dissection of the immune signals may help to stimulate intestinal adaptation, maintain function and prevent disease in premature infants, such as necrotizing enterocolitis (NEC). The group has established a unique model of collaboration among neonatal intensive care, pediatric gastroenterology and innate immunity. Fate and function of breast milk components (e.g. exosomes, microRNAs, proteins) with immuno-modulatory and anti-microbial properties are monitored both in the newborn gastrointestinal tract and mammary gland: passage, uptake, localization and distribution of breast milk bioactives in newborn animal GI tracts; and, immune responses of these milk constituents.
- Floris I, Kraft JD, Altosaar I. Roles of microRNA across prenatal and postnatal periods. Int. J. Mol. Sci. 2016, 17, 1994; doi:10.3390/ijms17121994 (12pp.)
- Altosaar et al., 2015. WO2015016829A1 Feed for lactating ruminants, A feed for ruminants may include at least one fatty acid component covalently linked with a carrier particle such that ingestion of the feed by lactating ruminants may provide for an increase in the amount of milk produced by the ruminant, and/or an increase in the fat content of the milk produced.
- Ward TL, Goto K, Altosaar I. 2014. Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice. Immunobiology 219(7): 537–546
- Ward TL, Spencer WJ, Davis LDR, Harrold JA, Mack DR, Altosaar I. 2014. Ingested soluble CD14 from milk is transferred intact into the blood of newborn rats. Pediatric Research 75(2): 252–258 doi:10.1038/pr.2013.225
- Ward TL, Hosid S, Ioshikhes I, Altosaar I. 2013. Human milk metagenome: a functional capacity analysis. BMC Microbiology 2013, 13:116-127 (25 May 2013 online) doi:10.1186/1471-2180-13-116. Designated "Highly accessed"
- Altosaar I, inventor. World Patent Appl. No. WO2013059907-A1 Detecting exposure of non-grain plant/plant part to plant pathogen, involves isolating starch granule from the non-grain, contacting with solvent to release peptide from surface of granule, and determining amino acid sequence of the peptide.
- Altosaar I et al. 2012 Funding decisions: Romania needs overseas reviewers (189 KB). Nature 492: 186(13 Dec.) doi:10.1038/492186c
- Zaidi M, El Bilali J, Koziol A, Ward T, Styles G, Greenham T, Faiella W, Son H, Wan S, Taga I, Altosaar I. 2012 Gene technology in agriculture, environment and biopharming: Beyond Bt-rice and building better breeding budgets for crops. J Plant Biochem & Biotechnology, 21 (Suppl 1):S2–S9.
- Fladung M, Altosaar I, Bartsch D, Baucher M, Boscaleri F, Gallardo F, Häggman H, Hoenicka H, Nielsen K, Paffetti D, Séguin A, Stotzky G, Vettori C. 2012. European discussion forum on transgenic tree biosafety (122 KB). Nature Biotechnology 30(1): 37-38.
- US Patent 7,214,862 Production of Human Granulocyte Macrophage-Colony Stimulating Factor in Plants. May 8, 2007.
Recent Supervised Theses
- Styles G, PhD 2015 Redesigning nature: Developing a more potent BMP2 molecule for expression in a transgenic puroindoline-rice expression system
- Ward TL, PhD 2014 Characterizing the immune-modulatory components of human milk: Fate and function of soluble CD14 and the human milk metagenome
- Koziol AG, PhD 2013 Application of direct-sequencing peptide proteomics to the characterization of antagonistic (endogenous and exogenous) proteins in cereal grains.
- Wan S, PhD 2012 Phytoremediation of nitrous oxide: Expression of nitrous oxide reductase from Pseudomonas stutzeriin transgenic plants and activity thereof.
- Wall ML, PhD 2011 The Starch Granule Surface: technological and biological implications of puroindoline and host-pathogen interactions.
Current Research Group
- Jamie Kraft – MSc student; Trevor Greenham - PhD student; Maryam Nourimand - Postdoctoral Fellow; Jordan Demone - Research Associate; Shen Wan, A.Erik Hansen - Senior Research Associates; Ib Taga – visiting scientist.