Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures.
Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures.
Ganapathi
T R
TR
Plant Cell culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Sunil Kumar
G B
GB
Srinivas
L
L
Revathi
C J
CJ
Bapat
V A
VA
D000911:Antibodies, Monoclonal; D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media; D004269:DNA, Bacterial; D018744:DNA, Plant / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D018745:Genome, Plant; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism*; D008353:Mannitol / Q000494:pharmacology; D010937:Plant Growth Regulators / Q000494:pharmacology; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013012:Sorbitol / Q000494:pharmacology; D013025:Glycine max / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D013997:Time Factors; D014170:Transformation, Genetic / Q000187:drug effects
D000911:Antibodies, Monoclonal; D003470:Culture Media; D004269:DNA, Bacterial; D018744:DNA, Plant; D006514:Hepatitis B Surface Antigens; D010937:Plant Growth Regulators; C036483:T-DNA; D008353:Mannitol; D013012:Sorbitol; D001374:Azacitidine