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

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