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Barcelona, Spain

Schmidt S.R.,ERA Biotech
Molecular Biotechnology | Year: 2013

Protein bodies are natural structures containing protein aggregates that exist in many organisms ranging from bacteria to mammals and plants. In bacteria they are often a phenomenon associated to over-expression of heterologous proteins. In mammals the so called Russell bodies indicate an accumulation of mutated immune globulins. In plants the protein bodies play a major role as protein storage organelle in seeds. Besides these natural cases, protein bodies can also be artificially induced primarily using self-assembling peptides. Frequently plant derived proteins such as prolamins or their derivatives are used. In some cases the help of an endoplasmatic retention signal is needed to create artificial protein bodies. The biotechnological application of protein bodies offers novel solutions such as the simplification of downstream processing in protein manufacture, the utilisation as particle for immunisation as vaccines or as carrier free self immobilised enzyme particle for many industrial catalytic processes. © 2012 Springer Science+Business Media, LLC. Source

Llop-Tous I.,Center for Research in Agricultural Genomics | Llop-Tous I.,ERA Biotech | Ortiz M.,Center for Research in Agricultural Genomics | Torrent M.,Center for Research in Agricultural Genomics | Ludevid M.D.,Center for Research in Agricultural Genomics
PLoS ONE | Year: 2011

Background: Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings: Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance: In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes. © 2011 Llop-Tous et al. Source

Madurga S.,University of Barcelona | Giralt E.,Institute Of Recerca Biomedica | Marzabal P.,ERA Biotech
Journal of Biological Chemistry | Year: 2010

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)8 of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys7 and Cys9) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

ERA Biotech | Date: 2012-09-28

The present invention relates generally to robust split inteins. The split inteins described herein are active over a large temperature range, including temperatures as low as 0 C., over a wide pH range, and in the presence of chaotropic salts. The split inteins also show high tolerance to sequence variability in fused heterologous polypeptides and therefore are useful in protein purification and engineering techniques.

ERA Biotech | Date: 2013-03-15

The present invention provides a pipette tip member with improved seal.

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