Transgenic Soybean Seed as Protein Expression System: Aqueous Extraction of Recombinant β-Glucuronidase

Robić, Goran; Farinas, Cristiane S.; Rech, E. L.; Miranda, Everson Alves

doi:10.1007/s12010-009-8637-5

Cited by 13 publications

(12 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This result is presented in Figure 5, which shows that seed proteins have acidic characteristics. This characteristic was also reported by Robic et al [38]. At the peptide level (Figure 6), 22% of all database peptides have a pI of less than 5, and 25% of them have a pI between 5 and 8.…”

Section: Resultssupporting

confidence: 85%

NanoUPLC-MSE proteomic data assessment of soybean seeds using the Uniprot database

Murad

Rech

2012

BMC Biotechnol

View full text Add to dashboard Cite

BackgroundRecombinant DNA technology has been extensively employed to generate a variety of products from genetically modified organisms (GMOs) over the last decade, and the development of technologies capable of analyzing these products is crucial to understanding gene expression patterns. Liquid chromatography coupled with mass spectrometry is a powerful tool for analyzing protein contents and possible expression modifications in GMOs. Specifically, the NanoUPLC-MSE technique provides rapid protein analyses of complex mixtures with supported steps for high sample throughput, identification and quantization using low sample quantities with outstanding repeatability. Here, we present an assessment of the peptide and protein identification and quantification of soybean seed EMBRAPA BR16 cultivar contents using NanoUPLC-MSE and provide a comparison to the theoretical tryptic digestion of soybean sequences from Uniprot database.ResultsThe NanoUPLC-MSE peptide analysis resulted in 3,400 identified peptides, 58% of which were identified to have no miscleavages. The experiment revealed that 13% of the peptides underwent in-source fragmentation, and 82% of the peptides were identified with a mass measurement accuracy of less than 5 ppm. More than 75% of the identified proteins have at least 10 matched peptides, 88% of the identified proteins have greater than 30% of coverage, and 87% of the identified proteins occur in all four replicates. 78% of the identified proteins correspond to all glycinin and beta-conglycinin chains.The theoretical Uniprot peptide database has 723,749 entries, and 548,336 peptides have molecular weights of greater than 500 Da. Seed proteins represent 0.86% of the protein database entries. At the peptide level, trypsin-digested seed proteins represent only 0.3% of the theoretical Uniprot peptide database. A total of 22% of all database peptides have a pI value of less than 5, and 25% of them have a pI value between 5 and 8. Based on the detection range of typical NanoUPLC-MSE experiments, i.e., 500 to 5000 Da, 64 proteins will not be identified.ConclusionsNanoUPLC-MSE experiments provide good protein coverage within a peptide error of 5 ppm and a wide MW detection range from 500 to 5000 Da. A second digestion enzyme should be used depending on the tissue or proteins to be analyzed. In the case of seed tissue, trypsin protein digestion results offer good databank coverage. The Uniprot database has many duplicate entries that may result in false protein homolog associations when using NanoUPLC-MSE analysis. The proteomic profile of the EMBRAPA BR-16 seed lacks certain described proteins relative to the profiles of transgenic soybeans reported in other works.

show abstract

Section: Resultssupporting

confidence: 85%

NanoUPLC-MSE proteomic data assessment of soybean seeds using the Uniprot database

Murad

Rech

2012

BMC Biotechnol

View full text Add to dashboard Cite

show abstract

“…Experiments are currently being run that focus on improving the TSP extraction and purification protocols, such as the separation of the hFIX from abundant seed endogenous proteins, by exploring different protein solubilities in a variety of extraction buffers, among other factors. This assumption is supported by the results of our recent studies evaluating downstream processing steps involved in the purification of b-glucuronidase from transgenic soybean seeds (Robić et al 2006(Robić et al , 2010. In addition, the results reported here on homozygous soybean lines expressing the hFIX protein and those from our studies on downstream processing of transgenic soybean seeds form a foundation for the further evaluation of transgenic soybean seeds expressing hFIX and their potential economic production.…”

Section: Discussionsupporting

confidence: 83%

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds

et al. 2010

Self Cite

View full text Add to dashboard Cite

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

show abstract

“…Such a theoretical scenario becomes immediately untenable if the soybean-derived therapeutic protein is destroyed following formulation from beans to a consumable. Recent success in our laboratory , and by others (Robic, Farinas, Rech, & Miranda, 2010), has demonstrated that a variety of formulations contain intact transgenic protein. While such empirical, stability studies will need to be performed with each particular transgenic protein, there are enumerable formulation strategies which have already been industrialized for soy consumables.…”

mentioning

confidence: 85%

Protein Expression Systems: Why Soybean Seeds?

Bost¹,

Piller²

2011

Soybean - Molecular Aspects of Breeding

View full text Add to dashboard Cite

Transgenic Soybean Seed as Protein Expression System: Aqueous Extraction of Recombinant β-Glucuronidase

Cited by 13 publications

References 24 publications

NanoUPLC-MSE proteomic data assessment of soybean seeds using the Uniprot database

NanoUPLC-MSE proteomic data assessment of soybean seeds using the Uniprot database

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds

Protein Expression Systems: Why Soybean Seeds?

Contact Info

Product

Resources

About