Accession Number:

AD1041051

Title:

Cell-Free Protein Synthesis from Genomically Recoded Bacteria Enables Multisite Incorporation of Noncanonical Amino Acids (Postprint)

Personal Author(s):

Corporate Author:

Northwestern University Evanston United States

Report Date:

2018-03-23

Abstract:

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 or - 30 mgL superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation or 98 and yield 96 or - 3 mgL. We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

Descriptive Note:

Journal Article - Open Access

Supplementary Note:

Nature Communications, 9, 01 Jan 0001, 01 Jan 0001,

Pages:

0012

Descriptors:

Identifiers:

Communities Of Interest:

Modernization Areas:

Distribution Statement:

Approved For Public Release;

Contract Number:

FA8650-15-D-5518

File Size:

1.48MB

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