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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 14, 2002 as doi:10.1096/fj.01-0363fje. |
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,3
* Institut für Biologie VII (Molekulare Biotechnologie, RWTH Aachen, 52074 Aachen, Germany; and
Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, 57392 Schmallenberg, Germany
2Correspondence: Institut für Biologie VII (Molekular Biotechnologie, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany. E-mail: vaquero{at}molbiotech.rwth-aachen.de
SPECIFIC AIMS
The aim of this work was to evaluate tobacco as a production system for a recombinant diabody derived from mAbT84.66 with potential use for diagnosis and treatment of colon cancer. We compared transient expression in agro-infiltrated leaves to stable expression in transgenic plants and the influence of targeting the recombinant protein to the apoplast and to the endoplasmic reticulum with respect to the accumulation of functional diabody. The feasibility of producing sufficient quantities of recombinant T84.66/GS8 diabody with the agro infiltration system for purification and biochemical analysis was also investigated.
PRINCIPAL FINDINGS
1. Expression of functional T84.66/GS8 diabody in agro-infiltrated leaves and transgenic plants
Recombinant T84.66/GS8 diabody genes were generated for targeting the protein to the endoplasmic reticulum (ER) and to the apoplast. Targeting to the secretory pathway was accomplished by using a plant codon-optimized amino-terminal signal peptide derived from a murine monoclonal antibody. ER retention was achieved using a carboxyl-terminal KDEL retrieval signal; the apoplast targeted diabody contained six carboxyl-terminal histidines (His6-tag). Expression of the diabody in tobacco leaves after vacuum-assisted infiltration with Agrobacteria (agro-infiltration) and in regenerated transgenic tobacco plants was analyzed and compared. Functional diabody targeted to either compartment was detected in protein extracts from agro-infiltrated tobacco leaves and transgenic tobacco plants by competition ELISA. Accumulation in agro-infiltrated leaves ranged between 1 and 5 mg/kg fresh weight for the apoplast and between 4 and 12 mg/kg for the ER targeted diabody. The highest accumulation in transgenic plants was 
0.9 mg/kg for the apoplast and 9 mg/kg for the ER targeted diabody. The rankings of the two constructs expressed in transgenic plants and agro-infiltrated leaves were the same, and the magnitude of accumulation of the recombinant protein in agro-infiltrated leaves and the best-expressing transgenic lines was similar.
2. Upscaling the agro infiltration system
To evaluate the agro-infiltration system for recombinant protein production and compare yields to transgenic plants, 1 kg of wild-type tobacco leaves was infiltrated. His6-tagged diabody was readily purified from agro-infiltrated tobacco leaves and from leaves of the highest expressing transgenic lines by immobilized metal ion affinity chromatography (IMAC). In both cases, sufficient amounts of the plant-expressed diabody were recovered to perform detailed biochemical analysis. Surprisingly, higher yields were obtained from agro-infiltrated leaves (1.5 mg/kg) than from the highest expressing transgenic plants (0.5 mg/kg).
3. Analysis of IMAC-purified T84.66/GS8 diabody from agro-infiltrated tobacco leaves
In reducing SDS-PAGE, the purified T84.66/GS8 diabody migrated with an electrophoretic mobility similar to the scFvT84.66 corresponding to 32 kDa and was recognized by His6 and T84.66-specific antibodies in Western blot. Minor amounts of degradation products were detected in Western blot and Coomassie-stained polyacrylamide gels and were slightly more pronounced in the preparation from transgenic plants. Initial studies using protease inhibitors indicated that these degradations did not occur in vitro during extraction and purification, but in vivo. No additional degradation in vitro was observed when the purified diabody was stored for 15 months at 4°C. In gel filtration, the IMAC-purified protein eluted as a single peak with a corresponding size of 67.5 kDa. Correct processing of the amino-terminal leader peptide of the recombinant diabody (preprotein) was confirmed by mass spectrometric analysis of a tryptic digest; preliminary analyses by nanospray mass spectrometry determined a molecular mass of 27206 Da, closely matching the expected mass of 27208 Da.
Functional diabody was detected in extracts from transgenic plants and infiltrated leaves as well as in the purified protein samples by competition ELISA. However, this method does not reveal the presence of nonfunctional molecules and so we investigated the activity of the IMAC-purified T84.66/GS8 diabody by electrophoretic mobility shift assay (EMSA) (Fig. 1
). Under nondenaturing conditions, the free diabody migrated as a single band (Fig. 1A
, lane 1). Addition of increasing amounts of recombinant CEA/NA3 to the diabody resulted in an increased fraction of shifted diabody due to antibody–antigen complex formation. In Western blot, two distinguishable shifted bands were detected that are likely to represent one CEA/NA3 molecule bound to one diabody molecule and two molecules of CEA/NA3 bound to one diabody molecule. In the presence of excess CEA/NA3, the band for free diabody and the intermediate band were undetectable (Fig. 1)
. This was confirmed by gel filtration, where in the presence of excess antigen the elution peak of free diabody disappeared completely and a new peak for the antibody–antigen complex appeared.
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The function of the purified protein was further investigated by immunofluorescence labeling of LS174 human colon carcinoma cells that express CEA on their cell surface. Fluorescence staining was observed after incubation with T84.66/GS8 diabody or T84.66 mAb, followed by reaction with anti-T84.66 mAb and detection with AlexaFluorTM568-conjugated goat anti-mouse-H+L-specific polyclonal antibodies. In contrast, incubation with an isotype-matched control mAb or CEA-negative HEK 293 cells incubated with T84.66/GS8 diabody were not stained (Fig. 2
). These results demonstrated that T84.66/GS8 diabody purified from tobacco not only recognized coated and soluble recombinant CEA/NA3, but also native, GPI-anchored CEA on the surface of LS174T cells (Fig. 2)
.
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CONCLUSIONS
This study demonstrates that tobacco is a suitable host for production of T84.66/GS8 diabody. Production and purification of His6-tagged diabody was successful not only from transgenic tobacco plants, but also from a scaled-up agro-infiltration system. Yields obtained from agro-infiltrated tobacco leaves were slightly higher, which might be due to a higher promotor activity and gene dosage during the transient expression.
The biochemical analyses of the one-step IMAC-purified protein showed that tobacco cells expressed and correctly processed the T84.66/GS8 diabody, and preliminary data from mass spectrometry suggested that post-translational modifications did not occur. Functionality of T84.66/GS8 diabody was shown by its ability to recognize recombinant CEA/NA3 produced in yeast and the membrane-anchored full-length CEA on CEA-expressing LS174T cells. The presence of nonfunctional molecules in the purified protein preparation, important for evaluating plant-based production systems, was investigated by EMSA and size exclusion HPLC. The absence of detectable inactive protein led us to conclude that only functional diabody was purified from tobacco cells and supports the suitability of tobacco as production system for T84.66/GS8 diabody.
The results of transient expression by agro infiltration correlated with those from stable expression in transgenic plants. This similarity is likely due to factors that influence accumulation of the recombinant protein in both systems, such as transcript and protein stability, efficiency of translation, folding, post-translational processing, and subcellular localization. The agro infiltration system is suitable not only for testing and scoring plant expression constructs and predicting their performance in transgenic plants, but also for purifying the recombinant protein. This facilitates faster and more detailed analysis of plant-expressed recombinant proteins than most plant-based expression systems permit. Results obtained from agro infiltration can then be considered for selecting constructs to establish transgenic plants for field production of the recombinant antibody.
In summary, our results show that T84.66/GS8 diabody was successfully expressed in tobacco and the purified protein was functional. However, the current accumulation levels are lower than those reported for other antibodies that have been expressed in tobacco and other plants. Why these levels were not achieved for T84.66/GS8 diabody is unclear, but there are preliminary indications that the stability of the mRNA could be a limiting factor. In agreement with earlier reports, higher accumulation of the recombinant antibody was observed within the ER. Although small peptide tags help target the recombinant protein to a favorable subcellular compartment or to ease purification, they generally are not desirable for therapeutic applications. Consequently, targeting and purification strategies that do not rely on peptide tags should be developed.
The development and application of antibody-based reagents in the clinic require robust expression systems allowing the production of pure and native recombinant protein in a fast, safe, and economical way. We believe that plants, particularly a combination of transient and stable expression systems, meet these demands as demonstrated in CEA-specific T84.66/GS8 diabody.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0363fje; to cite this article, use FASEB J. (January 14, 2002) 10.1096/fj.01-0363fje 
3 Current address: Beckman Research Institute of the City of Hope, Duarte, CA, USA.
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