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

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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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Abbreviations

VHH:

Variable domain of camelid heavy chain antibodies

ELISA:

Enzyme-linked immunosorbent assay

TSP:

Total soluble proteins

GUS:

β-Glucuronidase

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Acknowledgments

This work was supported by the grant AEGR 233231 INTA 2009-2011. EML is a fellow of Agencia Nacional de Promoción Científica y Técnológica (ANPCyT, Argentina). EFA is a fellow of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), FBA is research scientist of CONICET, and LG, VP and AW are research scientists of CONICET and Instituto Nacional de Tecnología Agropecuaria (INTA).

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Authors and Affiliations

  1. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), CONICET, Vuelta de Obligado 2490, C1428ADN, Buenos Aires, Argentina

    Ezequiel M. Lentz, E. Federico Alfano & Fernando F. Bravo-Almonacid

  2. Instituto de Virología, CICV y A, INTA, Castelar, Buenos Aires, Argentina

    Lorena Garaicoechea, Viviana Parreño & Andrés Wigdorovitz

  3. Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina

    Fernando F. Bravo-Almonacid

Authors
  1. Ezequiel M. Lentz
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  2. Lorena Garaicoechea
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  3. E. Federico Alfano
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  4. Viviana Parreño
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  5. Andrés Wigdorovitz
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  6. Fernando F. Bravo-Almonacid
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Corresponding author

Correspondence to Fernando F. Bravo-Almonacid.

Electronic supplementary material

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425_2012_1642_MOESM1_ESM.tif

Supplementary material 1. Phenotype of VHH transplastomic plants. a Heteroplasmic phenotype of a VHH plant that was subjected to five regeneration rounds in selective media and then transferred to soil in greenhouse conditions (right). A VP8* transplastomic plant (Lentz et al. 2011) grown in the same conditions is also shown for comparison (left). b Seeds from the plants shown in “a” were germinated in the presence of 500 mg/l spectinomycin. Wild-type seeds were included in the same plates as the control. 108 x 164 mm (300 x 300 DPI) (TIFF 7305 kb)

425_2012_1642_MOESM2_ESM.tif

Supplementary material 2. Preliminary molecular analysis of VHH and pep-VHH transplastomic plants. a Northern blot detection of plastidic transcripts containing the transgene sequence in VHH transplastomic plants. The position of 23S (2.9 kb) and 16S (1.5 kb) rRNA is indicated on the left. Ribosomal RNA 23S is shown as a reference of total RNA load. b Physical map of the chloroplast genome (WT ptDNA) at the insertion site and Southern blot analysis of pep-VHH independent lines (A, B, C and D) that were subjected to three (R3) or four (R4) regeneration rounds. Expected band sizes for WT and pep-VHH plants are 6.4 kb and 1.9 kb, respectively. 155 x 122 mm (300 x 300 DPI) (TIFF 2599 kb)

425_2012_1642_MOESM3_ESM.jpg

Supplementary material 3. Growth delay of a six-month-old pep-VHH transplastomic plant that produced transgenic offspring. A non-related transplastomic line is shown on the left for comparison (four months old). Seeds from the pep-VHH plant shown were germinated in the presence of 500 mg/l spectinomycin (sp). Wild-type seeds were included in the same plates as control. 454 x 218 mm (100 x 100 DPI) (JPEG 476 kb)

425_2012_1642_MOESM4_ESM.jpg

Supplementary material 4. Growth arrest and chlorotic leaf phenotype in a pep-VHH transplastomic line. Each line was grown in the shade (<30 μmol s-1 m-2, left), and in normal light (100–300 μmol s-1 m-2, right) in greenhouse. Pictures were taken at 10 and 14 weeks. A GUS-E-VHH, a wild-type and a non-related transplastomic plant (VP8*, Lentz et al. 2011) were included for comparison. 454 x 430 mm (100 x 100 DPI) (JPEG 1138 kb)

425_2012_1642_MOESM5_ESM.jpg

Supplementary material 5. Expression and quantification of GUS-E-VHH, in different leaves of a transplastomic plant. a 8% SDS-PAGE, stained with Coomassie brilliant blue and western blot using an antiserum against VHH. Lanes were loaded with total protein extracts obtained from 4 mg of fresh leaf tissue. b GUS-E-VHH quantification in leaf protein extracts by western blot in 15% gels, using purified VHH produced in E. coli as standard. Band intensities were quantified using the Image J program (NIH, http://rsbweb.nih.gov/ij) and are presented in Fig. 6. The position of VHH, GUS-E-VHH and the large subunit of RuBisCO (RbcL) are indicated. WT wild-type tobacco. c GUS-E-VHH transplastomic plant used in the study. 565 x 658 mm (100 x 100 DPI) (JPEG 1178 kb)

425_2012_1642_MOESM6_ESM.jpg

Supplementary material 6. Expression and quantification in different leaves of pep-VHH transplastomic plants grown under normal light and in the shade, and quantification. a Plants used in the study grown under normal light (100–300 μmol s-1 m-2) or in the shade (<30 μmol s-1 m-2). b 15% SDS-PAGE stained with Coomassie brilliant blue and western blot using an antiserum against VHH. Lanes were loaded with total protein extracts obtained from 4 mg of fresh leaf tissue. c VHH quantification in leaf protein extracts by western blot in 15% gels, using purified VHH produced in E. coli as standard. Band intensities were quantified using the Image J program (NIH, http://rsbweb.nih.gov/ij) and are presented in Fig. 7. The position of VHH and the large subunit of RuBisCO (Rb) are indicated. WT wild-type tobacco. 499 x 850 mm (100 x 100 DPI) (JPEG 1825 kb)

425_2012_1642_MOESM7_ESM.jpg

Supplementary material 7. Expression and quantification in different leaves of pep-VHH transplastomic plants grown under low light and in vitro in the shade, and quantification. a Plants used in the study grown under low light (30–100 μmol s-1 m-2) or in vitro in the shade (<30 μmol s-1 m-2). b 15% SDS-PAGE stained with Coomassie brilliant blue and western blot using an antiserum against VHH. Lanes were loaded with total protein extracts obtained from 4 mg of fresh leaf tissue. c VHH quantification in leaf protein extracts by western blot in 15% gels, using purified VHH produced in E. coli as standard. Band intensities were quantified using the Image J program (NIH, http://rsbweb.nih.gov/ij) and are presented in Fig. 7. The position of VHH and the large subunit of RuBisCO (RbcL) are indicated. 509 x 702 mm (100 x 100 DPI) (JPEG 1519 kb)

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Lentz, E.M., Garaicoechea, L., Alfano, E.F. et al. Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco. Planta 236, 703–714 (2012). https://doi.org/10.1007/s00425-012-1642-x

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