Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.
Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.
Department of Biochemistry, C.B.S.H., G. B. Pant University of Agriculture and Technology, Pantnagar, 263145, Uttarakhand, India, shef.dobhal@gmail.com.
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S
Dobhal
V K
VK
Chaudhary
A
A
Singh
D
D
Pandey
A
A
Kumar
S
S
Agrawal
D015153:Blotting, Western; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D005966:Glucuronidase