High yield recombinant silk-like protein production in transgenic plants through protein targeting

Abstract

DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a protein targeting approach to accumulate DP1B in apoplast, ER lumen, and vacuole in Arabidopsis cells, by utilizing appropriate combinations of sporamin-targeting determinant peptides and ER retention peptide. The approach has dramatically enhanced DP1B accumulation, resulting in high production yield. The accumulation can be as high as 8.5 and 6.7% total soluble protein in leaf tissue by targeting to apoplast and ER lumen, respectively, or as high as 18 and 8.2% total soluble protein in seeds by targeting to ER lumen and vacuole, respectively. However, the vacuole targeting in leaves and the apoplast targeting in seeds have failed to accumulate full length DP1B molecules or any DP1B at all, respectively, suggesting that they may not be suitable for applications in leaf tissues and seeds. Data in this study recommend a combination of seed-specific expression and ER-targeting as one of the best strategies for yield enhancement of plant-based DP1B production.

Recombinant Spider Silks—Biopolymers with Potential for Future Applications

“…The recombinant silk protein could easily be purified by a combination of heat treatment, acidification and ammonium sulfate precipitation [35,41]. In addition to tobacco and potatoes another engineered spidroin based on MaSp1 from Nephila clavipes was produced in the seeds of Arabidopsis, yielding 18% of the desired engineered spidroin within the total soluble protein, which makes this a viable means of protein production [42,43].…”

Transient Expression of an Immunogenic Envelope Attachment Glycoprotein of Nipah Virus in Nicotiana benthamiana

“…The ER contains a relatively low amount of proteolytic enzymes and the presence of molecular chaperones favours disulphide bond formation and correct protein folding, together with an oxidizing environment (Faye et al 2005;Nuttall et al 2002) which can improve heterologous protein stability. The positive impacts of ER retention have been documented for various recombinant proteins production, including antibodies (Stoger et al 2002), human interleukin-4 (Gomord et al 2004;Ma et al 2005), SARS corona virus S protein antigen (Pogrebnyak et al 2005), Aspergillus phytase (Peng et al 2006) and synthetic silk-like protein DR1B (Yang et al 2005).…”

High-level expression of sucrose inducible sweet potato sporamin gene promoter: &#x3b2;-glucuronidase fusion gene in transgenic <i>Nicotiana plumbaginifolia</i>

“…GUS protein (accession number, S69414) has one site which is glycosylated with N-glycosylation, and GUS activity has been decreased by N-glycosylation (Iturriaga et al 1989). Sporamin protein was transported to vacuoles by way of Golgi apparatus (Yang et al 2005) and was glycosylated during transportation (Shimizu et al 2005). Therefore, in our study it is possible that decrease of GUS activities were induced in the Sa, 3a, SE, 3E, Sv and 3v transformants because the GUS proteins had sorting sequences of sporamin and were glycosylated by sorting.…”