Performance benchmarking of four cell-free protein expression systems

Gagoski, Dejan, Polinkovsky, Mark E., Mureev, Sergey, Kunert, Anne, Johnston, Wayne, Gambin, Yann and Alexandrov, Kirill (2015) Performance benchmarking of four cell-free protein expression systems. Biotechnology and Bioengineering, 113 2: 292-300. doi:10.1002/bit.25814

Author Gagoski, Dejan
Polinkovsky, Mark E.
Mureev, Sergey
Kunert, Anne
Johnston, Wayne
Gambin, Yann
Alexandrov, Kirill
Title Performance benchmarking of four cell-free protein expression systems
Journal name Biotechnology and Bioengineering   Check publisher's open access policy
ISSN 1097-0290
Publication date 2015
Year available 2015
Sub-type Article (original research)
DOI 10.1002/bit.25814
Open Access Status Not Open Access
Volume 113
Issue 2
Start page 292
End page 300
Total pages 9
Place of publication Hoboken, NJ United States
Publisher John Wiley & Sons
Collection year 2016
Language eng
Formatted abstract
Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species—particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.
Keyword In vitro protein translation
Cell-free protein expression
Protein aggregation
E. coli CF
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
Institute for Molecular Bioscience - Publications
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Citation counts: TR Web of Science Citation Count  Cited 1 times in Thomson Reuters Web of Science Article | Citations
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Created: Thu, 03 Dec 2015, 08:49:15 EST by Susan Allen on behalf of Institute for Molecular Bioscience