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A Microscale Yeast Cell Disruption Technique for Integrated Process Development Strategies.
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- Author(s): Marc D. Wenger; Peter DePhillips; Daniel G. Bracewell
- Source:
Biotechnology Progress; Jun2008, Vol. 24 Issue 3, p606-614, 9p- Subject Terms:
- Source:
- Additional Information
- Abstract: Miniaturizing protein purification processes at the microliter scale (microscale) holds the promise of accelerating process development by enabling multi-parallel experimentation and automation. For intracellular proteins expressed in yeast, small-scale cell breakage methods capable of disrupting the rigid cell wall are needed that can match the protein release and contaminant profile of full-scale methods like homogenization, thereby enabling representative studies of subsequent downstream operations to be performed. In this study, a noncontact method known as adaptive focused acoustics (AFA) was optimized for the disruption of milligram quantities of yeast cells for the subsequent purification of recombinant human papillomavirus (HPV) virus-like particles (VLPs). AFA operates by delivering highly focused, computer-controlled acoustic radiation at frequencies significantly higher than those used in conventional sonication. With this method, the total soluble protein release was equivalent to that of laboratory-scale homogenization, and cell disruption was evident by light microscopy. The recovery of VLPs through a microscale chromatographic purification following AFA treatment was within 10% of that obtained using homogenization, with equivalent product purity. The addition of a yeast lytic enzyme prior to cell disruption reduced processing time by nearly 3-fold and further improved the comparability of the lysate to that of the laboratory-scale homogenate. In addition, unlike conventional sonication methods, sample heating was minimized (≤8 °C increase), even using the maximum power settings required for yeast cell disruption. This disruption technique in combination with microscale chromatographic methods for protein purification enables a strategy for the rapid process development of intracellularly expressed proteins. [ABSTRACT FROM AUTHOR]
- Abstract: Copyright of Biotechnology Progress is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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