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

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), CONICET, Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina.
Planta (Impact Factor: 3.26). 04/2012; 236(2):703-14. DOI: 10.1007/s00425-012-1642-x
Source: PubMed


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.

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Available from: Ezequiel Lentz, Sep 13, 2014
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    • "In tobacco chloroplasts, a peptide based therapy for type II diabetes was developed and delivered orally in mice fed tobacco accumulating extendin-4 (Kwon et al., 2013). Also, V H H domains against rotavirus were produced in tobacco chloroplasts at 2–3% total soluble protein, although they required translational fusions to stability domains or redirection to the lumen in order to accumulate to detectable levels (Lentz et al., 2012). In our study, the transgenic microalgae was non-photosynthetic due to the higher accumulation levels afforded by psbA knockouts in C. reinhardtii. "
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