1 research outputs found
Cell-free Protein Synthesis from a Release Factor 1 Deficient <i>Escherichia coli</i> Activates Efficient and Multiple Site-specific Nonstandard Amino Acid Incorporation
Site-specific incorporation of nonstandard
amino acids (NSAAs)
into proteins enables the creation of biopolymers, proteins, and enzymes
with new chemical properties, new structures, and new functions. To
achieve this, amber (TAG codon) suppression has been widely applied.
However, the suppression efficiency is limited due to the competition
with translation termination by release factor 1 (RF1), which leads
to truncated products. Recently, we constructed a genomically recoded <i>Escherichia coli</i> strain lacking RF1 where 13 occurrences
of the amber stop codon have been reassigned to the synonymous TAA
codon (<i>rEc.E13.ΔprfA</i>). Here, we assessed and
characterized cell-free protein synthesis (CFPS) in crude S30 cell
lysates derived from this strain. We observed the synthesis of 190
± 20 μg/mL of modified soluble superfolder green fluorescent
protein (sfGFP) containing a single <i>p</i>-propargyloxy-l-phenylalanine (pPaF) or <i>p</i>-acetyl-l-phenylalanine. As compared to the parent <i>rEc.E13</i> strain with RF1, this results in a modified sfGFP synthesis improvement
of more than 250%. Beyond introducing a single NSAA, we further demonstrated
benefits of CFPS from the RF1-deficient strains for incorporating
pPaF at two- and five-sites per sfGFP protein. Finally, we compared
our crude S30 extract system to the PURE translation system lacking
RF1. We observed that our S30 extract based approach is more cost-effective
and high yielding than the PURE translation system lacking RF1, ∼1000
times on a milligram protein produced/$ basis. Looking forward, using
RF1-deficient strains for extract-based CFPS will aid in the synthesis
of proteins and biopolymers with site-specifically incorporated NSAAs