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PEDS Advance Access published online on September 28, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn049
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Facile, reagentless and in situ release of Escherichia coli intracellular enzymes by heat-inducible autolytic vector for high-throughput screening

Zhen Cai1, Wanghui Xu1, Rui Xue2 and Zhanglin Lin1,3

1Department of Chemical Engineering 2Department of Chemistry, Tsinghua University, 1 Tsinghua Garden Road, Beijing 100084, China

3 To whom correspondence should be addressed. E-mail: zhanglinlin{at}mail.tsinghua.edu.cn

In an effect to broaden the application of the heat-inducible autolytic vector pUC18-cI857/pR-SRRz-rrnB previously developed, a new vector pUC18-cI857/pR(T41C)-SRRz-rrnB (pEAS-1b) was quantitatively characterized under various growth temperatures, heat induction temperatures and durations, and IPTG (isopropyl β-D-thiogalactoside) induction times, after resolving its erratic lysis profile found previously. Escherichia coli BL21 cells harboring this vector grew well at temperatures <36°C, and lysed efficiently (97.0 ± 0.8%) just 0.5 h after heat induction at 42°C for 30 min when cell growth was performed at 35°C. Application of this autolytic vector either in 96-well plates, or on nitrocellulose membranes, or on agar plates led to facile, efficient and consistent release of intracellular recombinant enzymes (e.g., a lysis efficiency of 91.8 ± 1.1% was obtained in 96-well plates). Further application in directed evolution was illustrated by improving the thermostability of amadoriase using this vector. This reagentless and in situ cell lysis method has the potentials for lysis of miniaturized samples in clinical diagnosis and bioanalytical detection, and even for lysis of cells in the microarray format.

Keywords: cell lysis/E.coli/heat-inducible autolytic vector/high-throughput screening

Received April 27, 2008; revised August 21, 2008; accepted August 22, 2008.


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