Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco

Abstract

Fragments from camelid single-chain antibodies known as VHHs or nanobodies represent a valuable tool in diagnostics, investigation and passive immunity therapy. Here, we explored different strategies to improve the accumulation of a neutralizing VHH antibody against rotavirus in tobacco transplastomic plants. First, we attempted to express the VHH in the chloroplast stroma and then two alternative strategies were carried out to improve the expression levels: expression as a translational fusion to the β-glucuronidase enzyme (GUS-E-VHH), and redirection of the VHH into the thylakoid lumen (pep-VHH). Every attempt to produce transplastomic plants expressing the VHH in the stroma was futile. The transgene turned out to be unstable and the presence of the VHH protein was almost undetectable. Although pep-VHH plants also presented some of the aforementioned problems, higher accumulation of the nanobody was observed (2-3% of the total soluble proteins). The use of β-glucuronidase as a partner protein turned out to be a successful strategy and expression levels reached 3% of the total soluble proteins. The functionality of the VHHs produced by pep-VHH and GUS-E-VHH plants was studied and compared with that of the antibody produced in Escherichia coli. This work contributes to optimizing the expression of VHH in transplastomic plants. Recombinant proteins could be obtained either by accumulation in the thylakoid lumen or as a fusion protein with β-glucuronidase, and both strategies allow for further optimization.

Plastid Proteostasis and Heterologous Protein Accumulation in Transplastomic Plants

“…Conversely, the same proteins when localized into the stroma are unable to form this type of covalent linkage. A similar conclusion has been drawn for a nanobody with intramolecular disulfide bonds expressed both in the stroma and in the thylakoid lumen (Lentz et al, 2012). This is not surprising, as the thylakoid lumen, as well as the intermembrane space of mitochondria, is topologically equivalent to the periplasmic space of bacteria, and active thiol oxidation pathways are present in these compartments in order to promote protein folding and regulate protein function (Herrmann et al, 2009).…”

Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts

“…The translational fusion to another protein that presents important stability has been previously used for nuclear transgenic plants rendering positive results [42][43][44], as well as for transplastomic plants [20,[45][46][47]. In this work, we chose the b-glucuronidase, a remarkably stable protein in the chloroplast stroma [48] which proved useful as a carrier to achieve high expression levels in transplastomic tobacco plants [49].…”

Molecular Farming: Hosting Plants as Green Factories

“…One of the major beneficial outcomes of this technology is genetically modified (GM, transgenic) plants in which one or several genes have been inserted into plant genome from various sources by using different means such as Agrobacterium tumefaciens or particle bombardment [1,2]. Organel genome of certain plant species also become as target to generate transplastomic plants [3,4]. Although GM plants have come along with a number of controvertial issues [1], the global planting area of GM plants constantly increased during last two decades and reached to 181.5 billion ha all around the world [5].…”