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.
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ABSTRACT: Since the serendipitous discovery 20 years ago of bona fide camelid heavy-chain antibodies, their single-domain antigen-binding fragments, known as VHHs or nanobodies, have received a progressively growing interest. As a result of the beneficial properties of these stable recombinant entities, they are currently highly valued proteins for multiple applications, including fundamental research, diagnostics, and therapeutics. Today, with the original patents expiring, even more academic and industrial groups are expected to explore innovative VHH applications. Here, we provide a thorough overview of novel implementations of VHHs as research and diagnostic tools, and of the recently evaluated production platforms for several VHHs and VHH-derived antibody formats.Trends in Biotechnology 04/2014; · 9.66 Impact Factor
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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
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ABSTRACT: In addition to conventional antibodies (with heavy and light chains), camelids also produce functional antibodies devoid of light chains (HCAbs) without the first constant domain (CH1). Their variable domains (VHH) have binding properties, high stability and solubility, and are considered the smallest available intact antigen–binding fragment derived from a functional immunoglobulin. For their practical utilities VHHs have been expressed in different platforms. This review aims to provide an update in the field of plant-made VHHs, their applications and limitations, and a discussion about the challenges for the near future in this field.Journal of Immunological Techniques in Infectious Diseases. 01/2014; 3(2):1-6.