Browsing by Author "Ritala, Anneli"

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In-solution antibody harvesting with a plant-produced hydrophobin-Protein A fusion
(2018-02) Kurppa, Katri; Reuter, Lauri J.; Ritala, Anneli; Linder, Markus B.; Joensuu, Jussi J.
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low-cost and scalable antibody capturing in solutions. Immunoglobulin-binding Protein A is widely used in affinity-based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two-phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35-fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB-induced protein body formation. We also demonstrated production of HFBI-Protein A fusion protein in tobacco BY-2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody-binding capacity of the Protein A block was similar to the nonfused Protein A. The best-performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two-phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.
Novel hydrophobin fusion tags for plant-produced fusion proteins
(2016-10-01) Reuter, Lauri; Ritala, Anneli; Linder, Markus; Joensuu, Jussi
A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
Hydrophobin fusion technology has been applied in the expression of several recombinant proteins in plants. Until now, the technology has relied exclusively on the Trichoderma reesei hydrophobin HFBI. We screened eight novel hydrophobin tags, T. reesei HFBII, HFBIII, HFBIV, HFBV, HFBVI and Fusarium verticillioides derived HYD3, HYD4 and HYD5, for production of fusion proteins in plants and purification by two-phase separation. To study the properties of the hydrophobins, we used N-terminal and C-terminal GFP as a fusion partner. Transient expression of the hydrophobin fusions in Nicotiana benthamiana revealed large variability in accumulation levels, which was also reflected in formation of protein bodies. In two-phase separations, only HFBII and HFBIV were able to concentrate GFP into the surfactant phase from a plant extract. The separation efficiency of both tags was comparable to HFBI. When the accumulation was tested side by side, HFBII-GFP gave a better yield than HFBI-GFP, while the yield of HFBIV-GFP remained lower. Thus we present here two alternatives for HFBI as functional fusion tags for plant-based protein production and first step purification.
Utilization of future food court side streams as alternative carbon and nutrient source for plant cell cultures
(2024-01-22) Lillberg, Amanda
Kemian tekniikan korkeakoulu | Master's thesis
Current food production capacity with only conventional agriculture is not sufficient to solely sustain the food demand for increasing human population. Sufficient food production could be enabled by combining conventional farming with cellular agriculture. Cellular agriculture refers to the production of agricultural commodities with plant, microbial and animal cells. Cellular agriculture, such as single cell protein synthesis, could also reduce the food production associated burden to land use and climate of food in the future. However, applications in cellular agriculture comes with many challenges, one of which includes the high costs for growth medium components. The aim of this thesis was to utilize alternative sources for carbon and nutrients for the plant cell growth medium to reduce costs. The alternative sources assessed in this thesis included biodegradable side streams from shopping centre environment. There, side streams accumulate from food courts and markets and could include orange peels, soda drink waste mix, expired bread, spent coffee grounds and brewer’s spent grains. Urine was also considered as a potential nutrient source since it possesses many valuable compounds that are required for the growth of plant cells. Future efficient purification technologies could permit the food regulation to allow the use of urine as a nutrient source. Plant cells require carbon and nutrients in a soluble form, therefore pre-treatments were applied for solid side streams to release monosaccharides from longer polysaccharide chains. The utilization potential of the side streams was evaluated by using them as culture medium components for cultivations of three non-taxonomically related plant cell lines: arctic bramble, tobacco BY-2, and barley. The side stream suitability was assessed by using biomass production after a cultivation period as a scale for comparison. Soda drink waste mix and synthetic urine contributed for the highest biomass concentrations, which was used as a measure for growth. These side streams were used as main components for optimization of growth media composition. Both side streams were successfully utilized for cultivations, especially for arctic bramble with notable increase in biomass concentration. Side streams were found to be potential culture medium components for plant cell cultures but notable differences in growth and growth behaviour between the studied plant cell lines were observed. The appliance of thus far unutilized side steams would enhance the circular economy and improve the food value chain. In addition, the economical sustainability of production would improve as costly components would be replaced with unutilized sources.
Utilization of industrial side streams as carbon and nutrient source for plant cell cultures
(2022-12-13) Pajumo, Maria
Kemian tekniikan korkeakoulu | Master's thesis
The nutrients and carbon sources used in cellular agriculture result in high continuous expenses which in turn slow down the interest of utilizing in plant cell culture -based products in food industry. Sucrose is the most used carbon source for plant cell cultures. It is also the most expensive single ingredient in plant cell culture medium. In industries, and especially in food production, large amounts of nutrient and sugar-rich side streams are generated, which the producers would like to valorize more effectively. The aim for this study was to evaluate the usability of industrial side streams as carbon and nutrient source for plant cell cultures. In the experiments the composition of nine different side stream samples S1 – S9 were analyzed and their usability to replace sucrose and nutrients in the traditional plant cell growth medium was evaluated. Based on the anal-yses, side streams S4 – S7 were determined the most suitable supplements for plant cell culture medium, of which S5 – S7 were used as a source of car-bon and side stream S4 as a nutrient source due to its high salt and nutrient content. Side stream supplementation was examined in suspension cultures of arctic bramble, lingonberry, silver birch, and tobacco. Based on previous studies, plant cells can utilize various mono- and disaccharides as a carbon source, but there are species differences in the utilization abilities. The effect of side stream supplementation on plant cell culture growth was assessed by comparing biomass, appearance, and viability of the cultures. The experiments confirmed that there are species-specific differences in the utilization ability of different carbohydrates. Arctic bramble cells were able to use side stream S6 as a carbon source and possibly adapt to utilize S7 after two cultivation cycles. Side streams S6 and S7 were found to increase lingonberry biomass production compared to the corresponding control, but the cells did not utilize the sugars contained in S7. In addition, the side stream S4 was successfully used as a source of potassium, sulfur, phosphorus, and nitrogen in arctic bramble culture medium. The supplementation even accelerated the cell growth and shortened the growth cycle from nine to seven days. The experiments conducted revealed that the side streams can be suc-cessfully used as nutrient and sucrose replacements in the plant cell culture medium. The replacements would in large scale significantly decrease the production costs of plant biomass and decrease the dependency on the tradi-tional carbon sources. The usage of side streams as a feedstock for cellular agriculture would also enhance effective material recycling in the food industry.