Process options in hepatitis B surface antigen extraction from transgenic potato

Belgin Dogan, Hugh Mason, Liz Richter, Jean B. Hunter, Michael L. Shuler

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined. The effects of temperature, the reducing agent β-mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined. Sedimentation profiles were performed to characterize HBsAg assembly into virus-like particles. Increasing the temperature of the sample for about 1 min increased the measured HBsAg antigenic activity. The optimum temperature was around 50 °C. A 3-fold enhancement of the antigenic activity was obtained in extract from transgenic potato expressing HBsAg, when monoclonal antibodies were used to assay for HBsAg. When antigenic activity was determined by polyclonal antibodies, no enhancement in the antigenic activity was obtained. Temperature may affect the conformation of the a epitope to which the monoclonal antibodies bind or alter the fluidity of surface lipid regions. BME increased the antigenic activity of HBsAg up to 4-fold when monoclonal antibodies directed against the a determinant were used, but there was no increase with polyclonal antibodies. This observation suggests that BME affects the structure or presentation of the a epitope. In the presence of BME and leupeptin, a proteinase inhibitor, higher antigenic activity was obtained. Leupeptin might protect the antigen, which might become more susceptible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases. Although both temperature and BME increased the antigenic activity of HBsAg individually, when combined their interaction was antagonistic, resulting in reduced antigenic activity. Different proteinase inhibitors, including leupeptin, aprotinin, E-64, pefabloc, and pepstatin, had no significant effect on HBsAg from potato extract in a 2 h period in the absence of BME. The sedimentation profile of potato-produced HBsAg was determined in 5-30% sucrose gradients. Yeast-derived recombinant HBsAg was used as a positive control. The HBsAg from transgenic potato showed sedimentation and density properties that are very similar to the yeast-produced antigen, indicating assembly into virus-like particles. BME treatment did not change the sedimentation profile.

Original languageEnglish (US)
Pages (from-to)435-441
Number of pages7
JournalBiotechnology Progress
Volume16
Issue number3
DOIs
StatePublished - 2000
Externally publishedYes

Fingerprint

Hepatitis B Surface Antigens
Solanum tuberosum
genetically modified organisms
potatoes
Peptide Hydrolases
proteinase inhibitors
Temperature
monoclonal antibodies
virus-like particles
Monoclonal Antibodies
temperature
polyclonal antibodies
Virion
epitopes
hepatitis B antigens
Epitopes
Yeasts
yeasts
antigens
Antigens

ASJC Scopus subject areas

  • Food Science
  • Biotechnology
  • Microbiology

Cite this

Process options in hepatitis B surface antigen extraction from transgenic potato. / Dogan, Belgin; Mason, Hugh; Richter, Liz; Hunter, Jean B.; Shuler, Michael L.

In: Biotechnology Progress, Vol. 16, No. 3, 2000, p. 435-441.

Research output: Contribution to journalArticle

Dogan, Belgin ; Mason, Hugh ; Richter, Liz ; Hunter, Jean B. ; Shuler, Michael L. / Process options in hepatitis B surface antigen extraction from transgenic potato. In: Biotechnology Progress. 2000 ; Vol. 16, No. 3. pp. 435-441.
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