Article

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: 3.35). 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.

0 0
 · 
0 Bookmarks
 · 
58 Views
  • [show abstract] [hide abstract]
    ABSTRACT: Nanobodies(®) (VHHs) provide powerful tools in therapeutic and biotechnological applications. Nevertheless, for some applications, bivalent antibodies perform much better, and for this, an Fc chain can be fused to the VHH domain, resulting in a bivalent homodimeric VHH-Fc complex. However, the production of bivalent antibodies in Escherichia coli is rather inefficient. Therefore, we compared the production of VHH7 and VHH7-Fc as antibodies of interest in Arabidopsis seeds for detecting prostate-specific antigen (PSA), a well-known biomarker for prostate cancer in the early stages of tumour development. The influence of the signal sequence (camel versus plant) and that of the Fc chain origin (human, mouse or pig) were evaluated. The accumulation levels of VHHs were very low, with a maximum of 0.13% VHH of total soluble protein (TSP) in homozygous T3 seeds, while VHH-Fc accumulation levels were at least 10- to 100-fold higher, with a maximum of 16.25% VHH-Fc of TSP. Both the camel and plant signal peptides were efficiently cleaved off and did not affect the accumulation levels. However, the Fc chain origin strongly affected the degree of proteolysis, but only had a slight influence on the accumulation level. Analysis of the mRNA levels suggested that the low amount of VHHs produced in Arabidopsis seeds was not due to a failure in transcription, but rather to translation inefficiency, protein instability and/or degradation. Most importantly, the plant-produced VHH7 and VHH7-Fc antibodies were functional in detecting PSA and could thus be used for diagnostic applications.
    Plant Biotechnology Journal 08/2013; · 6.28 Impact Factor
  • [show abstract] [hide abstract]
    ABSTRACT: Molecular farming is a technology that is very well suited to being applied in developing countries, given the reasonably high level of expertise in recombinant plant development in many centres. In addition, there is an urgent need for products such as inexpensive vaccines and therapeutics for livestock and for some human diseases-and especially those that do not occur or are rare in developed regions. South Africa and Argentina have been at the fore in this area among developing nations, as researchers have been able to use plants to produce experimental therapeutics such as nanoantibodies against rotavirus and vaccines against a wide variety of diseases, including Rabbit haemorrhagic disease virus, Foot and mouth disease virus, Bovine viral diarrhoea virus, bovine rotaviruses, Newcastle disease virus, rabbit and human papillomaviruses, Bluetongue virus, and Beak and feather disease virus of psittacines. A combination of fortuitous scientific expertise in both places, coupled with association with veterinary and human disease research centres, has enabled the growth of research groups that have managed to compete successfully with others in Europe and the USA and elsewhere, to advance this field. This review will cover relevant work from both South Africa and Argentina, as well as a discussion about the perspectives in this field for developing nations.
    Current pharmaceutical design 02/2013; · 4.41 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