Expression of synthetic human tropoelastin (hTE) protein inNicotiana tabacum

Abstract

ABSTRACT. Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal. Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium-mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5 th day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3-10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at »70 kDa from freshly extracted protein, while two degraded bands of »55 and »45 kDa, representing a pattern of tropoelastin were appeared with frozen samples. This study showed that biosynthetic ELN gene was successfully expressed into N. tabacum leaves using agroinfiltration technique.

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana

“…In addition, transient expression is genome integration-independent strategy, which will not be affected by position effects existing in stable transformation, once that the expression vector remains an episomal DNA molecule [22]. Our result is in accordance with previous works, which indicated that agroinfiltration could efficiently resulted in transferring of Agrobacterium into the plant leaves and robust expression of recombinant proteins [24,[52][53][54]. In our study, the infiltration of agrobacterium into the intercellular space of the leaf was performed with syringe method (Fig.…”

“…Process Biochemistry 70 (2018) [45][46][47][48][49][50][51][52][53][54] conditions consisting of 25°C/16-h light and 20°C/8-h dark and 40-65% relative humidity for four to six weeks. At this stage, the plant has at least six fully developed true leaves and no visible flower buds.…”

Elastin structure and its involvement in skin photoageing

“…Unlike other proteins, a single gene known as ELN, which is localized on chromosome 7 and expressed by different cell types throughout development, codes for elastin in humans [38,59,60]. As a result of alternative splicing, different isoforms of the soluble tropoelastin protein have previously been identified, suggesting a potential regulatory mechanism in the expression of this gene.…”

Advances in Plant-Derived Scaffold Proteins

“…A band corresponding to~70 kDa was detected during electrophoresis of fresh leaf extracts using an antibody against tropoelastin. However, recombinant tropoelastin degraded into two smaller molecular weight peptides when leaf extracts were frozen (Abdelghani et al, 2015). As the instability of protein going through freeze thaw of the leaf tissues will create a problem for potential harvesting and storage, recombinant tropoelastin directed to seeds may be more suitable, since recombinant protein can be stably stored in the seeds (Weichert et al, 2016).…”