Scripps VIVO scripps research logo

  • Index
  • Log in
  • Home
  • People
  • Organizations
  • Research
  • Events
Search form
As of April 1st VIVO Scientific Profiles will no longer updated for faculty, and the link to VIVO will be removed from the library website. Faculty profile pages will continue to be updated via Interfolio. VIVO will continue being used behind the scenes to update graduate student profiles. Please contact helplib@scripps.edu if you have questions.
How to download citations from VIVO | Alternative profile options

High-yield Escherichia coli-based cell-free expression of human proteins

Academic Article
uri icon
  • Overview
  • Research
  • Identity
  • Additional Document Info
  • View All
scroll to property group menus

Overview

authors

  • Michel, E.
  • Wuthrich, Kurt

publication date

  • May 2012

journal

  • Journal of Biomolecular NMR  Journal

abstract

  • Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [(15)N,(1)H]-HSQC NMR.

subject areas

  • Bacterial Proteins
  • Cell Extracts
  • Cell-Free System
  • Cloning, Molecular
  • Escherichia coli
  • Genetic Vectors
  • Humans
  • Isotope Labeling
  • Nuclear Magnetic Resonance, Biomolecular
  • Recombinant Fusion Proteins
scroll to property group menus

Research

keywords

  • Batch-mode cell-free protein expression
  • Escherichia coli S30 cell extract
  • Stable-isotope labeling
  • Structural biology of human proteins
scroll to property group menus

Identity

International Standard Serial Number (ISSN)

  • 0925-2738

Digital Object Identifier (DOI)

  • 10.1007/s10858-012-9619-4

PubMed ID

  • 22418693
scroll to property group menus

Additional Document Info

start page

  • 43

end page

  • 51

volume

  • 53

issue

  • 1

©2022 The Scripps Research Institute | Terms of Use | Powered by VIVO

  • About
  • Contact Us
  • Support