title: 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.
authors.affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), CONICET, Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina.
authors.forename: Ezequiel M
authors.initials: EM
authors.lastname: Lentz
authors.forename: Lorena
authors.initials: L
authors.lastname: Garaicoechea
authors.forename: E Federico
authors.initials: EF
authors.lastname: Alfano
authors.forename: Viviana
authors.initials: V
authors.lastname: Parreño
authors.forename: Andrés
authors.initials: A
authors.lastname: Wigdorovitz
authors.forename: Fernando F
authors.initials: FF
authors.lastname: Bravo-Almonacid
mesh_terms: D000818:Animals; D002161:Camelids, New World / Q000235:genetics* / Q000276:immunology; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D030821:Plants, Genetically Modified; D055550:Protein Stability; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*; D020524:Thylakoids / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes
chemical_list: D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D005966:Glucuronidase