ChemBioChem 2015-03-23

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Seok Hoon Hong, Yong-Chan Kwon, Rey W Martin, Benjamin J Des Soye, Alexandra M de Paz, Kirsten N Swonger, Ioanna Ntai, Neil L Kelleher, Michael C Jewett

Index: ChemBioChem. 16(5) , 844-53, (2015)

Full Text: HTML

Abstract

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-)), we synthesized 550±40 μg mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ∼1300 μg mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Related Compounds

Related Articles:

A survey of the interactome of Kaposi's sarcoma-associated herpesvirus ORF45 revealed its binding to viral ORF33 and cellular USP7, resulting in stabilization of ORF33 that is required for production of progeny viruses.

2015-05-01

[J. Virol. 89(9) , 4918-31, (2015)]

Aqueous-core PEG-coated PLA nanocapsules for an efficient entrapment of water soluble anticancer drugs and a smart therapeutic response.

2015-01-01

[Eur. J. Pharm. Biopharm. 89 , 30-9, (2015)]

SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression.

2015-01-01

[Nucleic Acids Res. 42(18) , 11433-46, (2014)]

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions.

2014-10-01

[Biochim. Biophys. Acta 1838(10) , 2615-24, (2014)]

The dual FAAH/MAGL inhibitor JZL195 has enhanced effects on endocannabinoid transmission and motor behavior in rats as compared to those of the MAGL inhibitor JZL184.

2014-09-01

[Pharmacol. Biochem. Behav. 124 , 153-9, (2014)]

More Articles...