Effect of Translation-Enhancing Nascent SKIK Peptide on the Arrest Peptides Containing Consecutive Proline

Ribosome arrest peptides
(RAPs) such as the SecM arrest peptide
(SecM AP: FSTPVWISQAQGIRAGP) and WPPP with consecutive Pro residues
are known to induce translational stalling in Escherichia
coli. We demonstrate that the translation-enhancing
SKIK peptide tag, which consists of four amino acid residues Ser-Lys-Ile-Lys,
effectively alleviates translational arrest caused by WPPP. Moreover,
the proximity between SKIK and WPPP significantly influences the extent
of this alleviation, observed in both PURE cell-free protein synthesis
and in vivo protein production systems, resulting in a substantial
increase in the yield of proteins containing such RAPs. Furthermore,
we unveil that nascent SKIK peptide tag and translation elongation
factor P (EF-P) alleviate ribosome stalling in consecutive-Pro-rich
protein to synergistically promote translation. A kinetic analysis
based on the generation of superfolder green fluorescent protein under
in vitro translation reaction reveals that the ribosome turnover is
enhanced by more than 10-fold when the SKIK peptide tag is positioned
immediately upstream of the SecM AP sequence. Our findings provide
valuable insights into optimizing protein production processes, which
are essential for advancing synthetic biology applications.