Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco

Geada, Déborah; Valdés, Rodolfo; Escobar, Arturo; Ares, Dulce; Torres, Edel; Blanco, Reinaldo; Ferro, Williams; Dorta, Dayamí; González, Marcos; Alemán, Marı́a Remedios; Padilla, Sigifredo; Gómez, Leonardo D.; Castillo, Norma del; Mendoza, Otto; Urquiza, Dioslaida; Soria, Yordanka; Brito, José A.; Leyva, Alejandro; Borroto, Carlos; Gavilondo, Jorge V.

doi:10.1016/j.biologicals.2007.02.007

Cited by 6 publications

(4 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…No significant differences were observed between bottom and top leaves, nor did temperature appear to make an important difference. TSP represents a mixture of proteins throughout the plant cell, but at least 50% of the TSP comprises RUBISCO (Geada et al 2007) which is compartmentalised in the relatively stable environment of the chloroplast and not exposed to the same array of proteases encountered by secreted IgG. It is interesting that CV-N yields were affected differently by alterations in temperature, but this may also be explained on the basis of protein stability.…”

Section: Discussionmentioning

confidence: 94%

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

et al. 2009

View full text Add to dashboard Cite

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.

show abstract

Section: Discussionmentioning

confidence: 94%

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

et al. 2009

View full text Add to dashboard Cite

show abstract

“…The main contaminant appears to be the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase represented under denatured conditions by its large (~ 53 kDa) and small (~15 kDa) subunits. The protein is known to be a major obstacle in achieving homogenous preparations of plant-expressed biopharmaceuticals [37]. …”

Section: Resultsmentioning

confidence: 99%

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

et al. 2010

View full text Add to dashboard Cite

BackgroundMucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.ResultsThe present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.ConclusionPlant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.

show abstract

“…Ammonium sulphate precipitation and extraction at low pH are particularly useful in green tissue extracts to precipitate contaminants such as cellular debris and photosynthetic pigments. These procedures are also useful to precipitate the highly abundant enzyme ribulose 1,5‐bisphosphate carboxylase oxygenase (RuBisCO) (Bendandi et al ., ; Lai et al ., ; Peckham et al ., ; Woodard et al ., ), which represents up to 50% of total soluble proteins (TSP) in leaves (Ellis, ) and often complicates the preparation of a highly purified protein product (Buyel et al ., ; Gaeda et al ., ; Ross and Zhang, ). On the other hand, ammonium sulphate precipitation is hardly adaptable to large‐scale production schemes and low pH extraction is not compatible with most pH‐sensitive protein products.…”

Section: Introductionmentioning

confidence: 99%

Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein

Robert

Goulet

D’Aoust

et al. 2015

Plant Biotechnology Journal

View full text Add to dashboard Cite

SummaryA key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein upregulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 lg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 lg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins.

show abstract

Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco

Cited by 6 publications

References 18 publications

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein

Contact Info

Product

Resources

About