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: 3.38). 04/2012; 236(2):703-14. DOI: 10.1007/s00425-012-1642-x
Source: PubMed

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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Plants have been proved as a novel production platform for a wide range of biologically important compounds such as enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. In this context, plastid genetic engineering can be potentially used to produce recombinant proteins. However, several challenges still remain to be overcome if the full potential of plastid transformation technology is to be realized. They include the development of plastid transformation systems for species other than tobacco, the expression of transgenes in non-green plastids, the increase of protein accumulation and the appearance of pleiotropic effects. In this paper, we discuss the novel tools recently developed to overcome some limitations of chloroplast transformation.
    Bioengineered. 11/2012; 3(6):329-33.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The plastid genome represents an attractive target of genetic engineering in crop plants. Plastid transgenes often give high expression levels, can be stacked in operons and are largely excluded from pollen transmission. Recent research has greatly expanded our toolbox for plastid genome engineering and many new proof-of-principle applications have highlighted the enormous potential of the transplastomic technology in both crop improvement and the development of plants as bioreactors for the sustainable and cost-effective production of biopharmaceuticals, enzymes and raw materials for the chemical industry. This review describes recent technological advances with plastid transformation in seed plants. It focuses on novel tools for plastid genome engineering and transgene expression and summarizes progress with harnessing the potential of plastid transformation in biotechnology.
    Current opinion in biotechnology 04/2014; 26C:7-13. · 7.82 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Since the first demonstration of stable transgene integration in the plastid genome (plastome) of higher plants, plastid transformation has been used for a wide range of purposes, including basic studies as well as biotechnological applications, showing that transplastomic plants are an effective system to produce recombinant proteins. Compared to nuclear transformation, the main advantages of this technology are the high and stable production level of proteins as well as the natural containment of transgenes. To date, more than 100 transgenes have been successfully expressed in plant chloroplasts. In some cases, however, unintended pleiotropic effects on plant growth and physiology were shown in transplastomic plants. In this paper, we review such effects and discuss some of the technologies developed to overcome them.
    Biotechnology Letters 10/2013; · 1.85 Impact Factor


Available from
Sep 13, 2014