Enhanced kinetic extraction of parvovirus B19 structural proteins

• Published in: Biotechnology and bioengineering 2002-11, Vol.80 (3), p.250-256
• Main Authors: Sico, Colleen, White, Stephen, Tsao, Eric, Varma, Amit
• Format: Article
• Language: English
• Subjects: Animals; B19; Baculoviridae - chemistry; Biological and medical sciences; Biotechnology; Capsid - chemistry; Capsid Proteins - isolation & purification; Cells, Cultured; extraction; Fundamental and applied biological sciences. Psychology; Gene Expression; Health. Pharmaceutical industry; Humans; Hydrogen-Ion Concentration; Industrial applications and implications. Economical aspects; Insecta - cytology; Insecta - virology; parvovirus; Production of active biomolecules; Quality Control; Recombinant Proteins - isolation & purification; Reproducibility of Results; Sensitivity and Specificity; Temperature; Vaccins; virus-like particle (VLP)
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.10509

Recombinant structural proteins (VP1 and VP2) of the human parvovirus B19 have been expressed simultaneously using the baculovirus expression system to form virus‐like particles (VLPs) that have potential use as vaccines. In this study, we report optimization of extraction conditions to recover these VLPs from cell paste. Under hypotonic conditions with neutral pH these VLPs were poorly extracted (up to 3% extraction). Addition of reducing agents, detergents, salts, and sonication did not improve the extractability. While screening for conditions to improve the extractability of the VLPs, we discovered that a combination of higher pH and elevated processing temperature significantly increased the extraction. Whereas increasing pH alone increased extractability from 3% to 6% (pH increased from 8.0 to 9.5), the effect of elevated temperature was much more substantial. At 50°C, we observed the extraction to be more than fivefold higher than that at room temperature (up to 25% extracted at pH 9.0). The kinetics of extraction at elevated temperatures showed a rapid initial rate of extraction (on the order of minutes) followed by a plateau. In addition, we compared the extraction of VP1 expressed alone. VP1 expressed alone is incapable of forming VLPs. We observed that non‐VLP VP1 was easily extractable (up to 60% extracted) under conditions in which the VP1 + VP2 VLPs were not extractable. From these studies we conclude that parvovirus B19 structural proteins expressed to form VLPs have a hindered extractability as compared with non‐VLP protein. This hindrance to extraction can be significantly reduced by processing at elevated temperatures and an increased pH, possibly due to the enhanced rates of solubilization and diffusion. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 250–256, 2002.