RESUMEN
We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.
Asunto(s)
Ingeniería Genética/métodos , Nicotiana/genética , Plantas Modificadas Genéticamente , Inhibidores de Proteasas/metabolismo , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismo , TransgenesRESUMEN
BACKGROUND: Recent studies have reported agronomically useful ectopic effects for recombinant protease inhibitors expressed in leaves of transgenic plants, including improved tolerance to abiotic stress conditions and partial resistance to necrotrophic pathogens. Here we assessed the effects of these proteins on the post-dormancy sprouting of storage organs, using as a model potato tubers expressing cysteine protease inhibitors of the cystatin protein superfamily. RESULTS: Sprout emergence and distribution, soluble proteins, starch and soluble sugars were monitored in tubers of cereal cystatin-expressing clones stored for several months at 4 °C. Cystatin expression had a strong repressing effect on sprout growth, associated with an apparent loss of apical dominance and an increased number of small buds at the skin surface. Soluble protein content remained high for up to 48 weeks in cystatin-expressing tubers compared to control (untransformed) tubers, likely explained by a significant stabilization of the major storage protein patatin, decreased hydrolysis of the endogenous protease inhibitor multicystatin and low cystatin-sensitive cysteine protease activity in tuber tissue. Starch content decreased after several months in cystatin-expressing tubers but remained higher than in control tubers, unlike sucrose showing a slower accumulation in the transgenics. Plantlet emergence, storage protein processing and height of growing plants showed similar time-course patterns for control and transgenic tubers, except for a systematic delay of 2 or 3 d in the latter group likely due to limited sprout size at sowing. CONCLUSIONS: Our data point overall to the onset of metabolic interference effects for cereal cystatins in sprouting potato tubers. They suggest, in practice, the potential of endogenous cysteine proteases as relevant targets for the development of potato varieties with longer storage capabilities.
Asunto(s)
Cistatinas/genética , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Proteínas de Plantas/genética , Solanum tuberosum/genética , Zea mays/genética , Cistatinas/metabolismo , Germinación , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/genética , Tubérculos de la Planta/crecimiento & desarrollo , Tubérculos de la Planta/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/metabolismo , Zea mays/metabolismoRESUMEN
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.
Asunto(s)
Inmunoglobulina G/genética , Nicotiana/genética , Hojas de la Planta/genética , Planticuerpos/genética , Proteolisis , Proteínas Recombinantes/genética , Vectores Genéticos , Humanos , Solanum lycopersicum/metabolismo , Hojas de la Planta/metabolismo , Planticuerpos/metabolismo , Inhibidores de Proteasas/metabolismo , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismoRESUMEN
Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by â¼45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway.
Asunto(s)
Péptido Hidrolasas/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/enzimología , Proteínas de Plantas/metabolismo , Agrobacterium/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Arabidopsis/enzimología , Arabidopsis/genética , Genoma de Planta/genética , Malato Deshidrogenasa/metabolismo , Ratones , Oryza/enzimología , Oryza/genética , Péptido Hidrolasas/genética , Péptido Hidrolasas/inmunología , Hojas de la Planta/efectos de los fármacos , Proteínas de Plantas/genética , Inhibidores de Proteasas/metabolismo , Estabilidad Proteica , Transporte de Proteínas , Proteolisis/efectos de los fármacos , Proteoma/metabolismo , Solanaceae/enzimología , Solanaceae/genética , Especificidad de la Especie , Nicotiana/citología , Nicotiana/efectos de los fármacos , Nicotiana/enzimología , Nicotiana/microbiología , TransfecciónRESUMEN
This study was undertaken to develop new probiotic products based on liquid maple sap or its concentrate. Sap and concentrate, with or without inulin (2%) were inoculated with Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus GG valio at initial counts of 107-108 CFU/ml. Viability was assessed over four weeks of storage at 4 °C and under in vitro simulated gastrointestinal conditions using dynamic gastrointestinal model known as TIM-1. Viability was maintained throughout the storage period at the same order of 107 to 108 CFU/ml. Inulin significantly enhanced the survivability during passage through the gastrointestinal tract simulator. The developed products could be an excellent alternative for delivering probiotics, especially for individuals suffering from lactose intolerance to dairy products.
Asunto(s)
Acer , Bifidobacterium/aislamiento & purificación , Tracto Gastrointestinal/microbiología , Lacticaseibacillus rhamnosus/aislamiento & purificación , Probióticos , Recuento de Colonia MicrobianaRESUMEN
Recombinant protease inhibitors represent useful tools for the development of insect-resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad-spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI-expressing and control (comparator) lines, followed by a two-dimensional gel electrophoresis (2-DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI-expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2-DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2-DE showed unaltered deposition patterns in SlCDI-expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI-expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel-generation transgenic plants.
Asunto(s)
Péptidos/genética , Proteínas de Plantas/genética , Tubérculos de la Planta/metabolismo , Solanum lycopersicum/genética , Solanum tuberosum/metabolismo , Electroforesis en Gel Bidimensional , Modelos Moleculares , Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Estructura Terciaria de Proteína , Proteoma/metabolismo , Solanum tuberosum/genética , TransgenesRESUMEN
We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in-built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359-368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI-expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%-40% depending on the light regime. Out of approximately 700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were up-regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase-PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two- to threefold in SlCDI-expressing lines. These observations, along with the unrelated biological functions assigned to MS-identified proteins up-regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human alpha(1)-antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by approximately 2.5-fold in SlCDI-expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.
Asunto(s)
Proteínas de Plantas/metabolismo , Inhibidores de Proteasas/metabolismo , Proteínas Recombinantes/metabolismo , Solanum tuberosum/metabolismo , Citosol/metabolismo , Electroforesis en Gel Bidimensional , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteoma/metabolismo , Proteínas Recombinantes/genética , Solanum tuberosum/genética , Transgenes , alfa 1-Antiquimotripsina/genética , alfa 1-Antiquimotripsina/metabolismoRESUMEN
Current screening and event-specific polymerase chain reaction (PCR) assays for the detection and identification of genetically modified organisms (GMOs) in samples of unknown composition or for the detection of non-regulated GMOs have limitations, and alternative approaches are required. A transgenic DNA fingerprinting methodology using restriction enzyme digestion, adaptor ligation, and nested PCR was developed where individual GMOs are distinguished by the characteristic fingerprint pattern of the fragments generated. The inter-laboratory reproducibility of the amplified fragment sizes using different capillary electrophoresis platforms was compared, and reproducible patterns were obtained with an average difference in fragment size of 2.4 bp. DNA insert fingerprints for 12 different maize events, including two maize hybrids and one soy event, were generated that reflected the composition of the transgenic DNA constructs. Once produced, the fingerprint profiles were added to a database which can be readily exchanged and shared between laboratories. This approach should facilitate the process of GMO identification and characterization.
Asunto(s)
Dermatoglifia del ADN/métodos , Glycine max/genética , Plantas Modificadas Genéticamente/genética , Zea mays/genética , Secuencia de Bases , ADN de Plantas/química , ADN de Plantas/genética , Ingeniería Genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/química , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Glycine max/química , Zea mays/químicaRESUMEN
Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.