Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli .

Journal Article (Journal Article)

Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly, protein extraction methods are predominantly restricted to chemical methods, and mechanical methods reliant on expensive specialized equipment more tuned for large-scale applications. In this article, we outline detailed protocols for the use of an engineered autolysis/autohydrolysis E. coli strain, in two-stage fermentations in shake-flasks. This two-stage fermentation protocol does not require optimization of expression conditions and results in high protein titers. Cell lysis in an engineered strain is tightly controlled and only triggered post-culture by addition of a 0.1% detergent solution. Upon cell lysis, a nuclease digests contaminating host oligonucleotides, which facilitates sample handling. This method has been validated for use at different scales, from microtiter plates to instrumented bioreactors. Graphic abstract: Two-stage protein expression, cell autolysis and DNA/RNA autohydrolysis. Reprinted with permission from Menacho-Melgar et al. (2020a). Copyright 2020 John Wiley and Sons.

Full Text

Duke Authors

Cited Authors

  • Menacho-Melgar, R; Lynch, MD

Published Date

  • January 20, 2022

Published In

Volume / Issue

  • 12 / 2

Start / End Page

  • e4297 -

PubMed ID

  • 35127987

Pubmed Central ID

  • PMC8799905

Electronic International Standard Serial Number (EISSN)

  • 2331-8325

International Standard Serial Number (ISSN)

  • 2331-8325

Digital Object Identifier (DOI)

  • 10.21769/bioprotoc.4297

Language

  • eng