Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Khalf, Moustafa; Goulet, Charles; Vorster, Juan; Anguenot, RaphaÃ«l; Fliss, I.; Michaud, Dominique

doi:10.1111/j.1467-7652.2009.00471.x

Cited by 35 publications

(25 citation statements)

References 77 publications

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“…Whole-leaf proteins were extracted in 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl and a commercial protease inhibitor cocktail (Roche, Laval QC, Canada) by disrupting tissue with tungsten carbide beads (QIAGEN, Mississauga ON, Canada) in a Mini-Beadbeater apparatus (BioSpec, Bartlesville OK, USA). Sl CDI was detected with commissioned polyclonal IgY antibodies raised in chicken against a hydrophilic surface loop of the protein [39] (Agrisera, Vännäs, Sweden) and alkaline phosphatase-conjugated rabbit anti-chicken IgY secondary antibodies (Sigma-Aldrich). Sl CYS8 was detected with commissioned polyclonal IgG antibodies raised in rabbit against bacterially expressed Sl CYS8 [35] (Agrisera) and alkaline phosphatase-conjugated goat anti-rabbit IgG as secondary antibodies (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

et al. 2013

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The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ∼3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.

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Section: Methodsmentioning

confidence: 99%

Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

et al. 2013

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“…A two-dimensional gel electrophoresis (2DE) based proteomic approach was used to compare protein profiles of transgenic Arabidopsis (Ren et al 2009;Ruebelt et al 2006), tomato (Corpillo et al 2004), potato (Khalf et al 2010;Lehesranta et al 2005) and maize (Barros et al 2010;Zolla et al 2008) lines and their non-GM counterparts. These studies revealed low percentages of proteins with significantly altered levels in transgenic and non-GM lines, and the differences in spot quantities either were part of the intended effects or fell within the range of natural variability.…”

Section: Introductionmentioning

confidence: 99%

Proteomic analysis of MON810 and comparable non-GM maize varieties grown in agricultural fields

et al. 2010

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Worldwide maize is the second major agricultural commodity and around one-fourth is currently biotech, with significant application of the insect resistant event MON810 particularly in the European Union. Grains are the major commercialized part of the plant, and can be harvested after maturity (for food and feed purposes) or at late milky-starchy stage (for forage uses, with the whole plant). We assessed possible proteomic unintended effects of the MON810 transgene using two-dimensional gel electrophoresis coupled to mass spectrometry. To keep in a realistic scenario we used plants grown in agricultural fields in a region where ~50% of maize was MON810, and analyzed grains at milky-starchy stage. In maize, differential transcripts and metabolites between GM and comparable non-GM varieties tend to be variety specific. Thus, we analyzed two variety pairs, DKC6575/Tietar and PR33P67/PR33P66 which are considered representative of Food and Agriculture Organization 700 and 600 varieties commercially grown in the region. MON810 and non-GM milky-starchy grains had virtually identical proteomic patterns, with a very small number of spots showing fold-variations in the 1-1.8 range. They were all variety specific and had divergent identities and functions. Although 2DE allows the analysis of a limited dataset our results support substantial equivalence between MON810 and comparable non-GM varieties.

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“…1b, IgE in the serum of every patient bound to a 42 kDa protein, i.e., a protein with the same molecular weight as that of patatin, a major allergen of potato. Other proteins that bound to the sera of potato-allergic patients had molecular weights of 14 kDa (subject 13), 21 kDa (subjects 1,5,7,8,10,11,14), and 75 kDa (subject 4). There were no clear differences in the patterns of IgE-binding proteins between non-transgenic potatoes and transgenic potatoes.…”

Section: Detection Of Ige-binding Proteins In Potatoes By 1d-immunoblmentioning

confidence: 99%

“…Various transgenic potatoes have been developed and their food safety has been assessed using serological and proteomic techniques. [9][10][11] Several cases of potato allergies have been reported, [12][13][14][15][16] and a major potato allergen that causes hypersensitive reactions has been identified as patatin (Sola t 1). 17) Potato allergy is considered to be cross-reactive with latex or pollens, and their cross-reactive fruit and vegetables.…”

mentioning

confidence: 99%

Immunoproteomic and Two-Dimensional Difference Gel Electrophoresis Analysis of Arabidopsis Dehydration Response Element-Binding Protein 1A (DREB1A)-Transgenic Potato

Nakamura¹,

Satoh²,

Nakamura³

et al. 2010

Biological & Pharmaceutical Bulletin

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To increase the quality and yield of crops, transgenic plants have been developed that show enhanced resistance to insects or herbicides, or improved growth and nutrient status. In addition, transgenic plants expressing proteins associated with stress responses are being developed in an effort to produce crops with greater resistance to stresses such as cold, heat, and salt.1) Genetic engineering of a transcriptional factor may affect expressions of multiple genes, therefore, there may be unintended effects on protein expression levels in transgenic plants. For appropriate safety assessment, therefore, it is important to evaluate the proteomics of transgenic plants, particularly with respect to allergenicity.Even after the establishment of the 2003 Codex guidelines for methods to assess safety of foods derived from transgenic organisms, there is still controversy regarding methods used to test their allergenicity.2) Allergenicity of foods derived from transgenic organisms has been evaluated using immunological methods such as immunoblots, histamine release assays, skin prick tests, and oral exposure. 3,4) Immunoproteomic analysis of allergens with sera from allergic patients is a useful method for comprehensively comparing unknown allergens as well as known allergens in extracts of foods derived from transgenic organisms. Previously, we reported application of the immunoproteome to assess allergenicities of GH-1 (growth hormone 1)-transgenic amago salmon 5) and enhanced green fluorescent protein (EGFP)-transgenic chicken meat.6) The two-dimensional difference gel electrophoresis (2D-DIGE) method, a gel-based proteomic technology, is useful for evaluating relative differences in protein expression, if any, between non-transgenic and transgenic crops. The CyDye DIGE fluors used to label samples are spectrally resolvable, size-and charge-matched fluorescent dyes. 7,8) The method enables analysis of one or more samples on the same gel and is highly sensitive, and has a wide range of detection.Potato (Solanum tuberosum) is a staple food that is consumed worldwide. Various transgenic potatoes have been developed and their food safety has been assessed using serological and proteomic techniques.9-11) Several cases of potato allergies have been reported, 12-16) and a major potato allergen that causes hypersensitive reactions has been identified as patatin (Sola t 1).17) Potato allergy is considered to be cross-reactive with latex or pollens, and their cross-reactive fruit and vegetables. [18][19][20] Other potato allergens, cathepsin D-, cysteine-, and serine protease inhibitors, all of which are members of the trypsin inhibitor family, have been designated as Sola t 2, Sola t 3, and Sola t 4, respectively. 21) These allergens make up approximately 70-80% of total soluble potato proteins. 22)In this study, we investigated the allergenicity of stresstolerant transgenic potatoes 23,24) To produce crops that are more tolerant to stresses such as heat, cold, and salt, transgenic plants have been produced those express stre...

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Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Cited by 35 publications

References 77 publications

Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

Proteomic analysis of MON810 and comparable non-GM maize varieties grown in agricultural fields

Immunoproteomic and Two-Dimensional Difference Gel Electrophoresis Analysis of Arabidopsis Dehydration Response Element-Binding Protein 1A (DREB1A)-Transgenic Potato

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