Presentation Information
[P03-365]Kinetic Analysis and Application of Translation-Enhancing Peptide for Protein Production
○Akimichi Yoshino1, Yuma Nishikawa1, Hideo Nakano1, Teruyo Kato1 (1. Grad. Sch. Bioagricultural Sci., Nagoya University (Japan))
Keywords:
SKIK peptide,polyproline,translation,cell-free protein synthesis,difficult-to-express protein
[Purpose]
Protein production is crucial for efficient biomanufacturing. However, ribosomal stalling, or translational arrest, induced by polyproline stretches or arrest peptides (APs) is considered one of the bottlenecks in the translation step, often leading to severely reduced yields. Our research group has developed a short translation-enhancing peptide, the SKIK tag (Ser-Lys-Ile-Lys), which promotes translation and increases protein production in Escherichia coli when fused to the N-terminus of target proteins. In addition, SKIK has been shown to alleviate AP-induced translational arrest. However, the detailed mechanism underlying its effect remains unknown. In this study, we aimed to get insights into the translation-enhancing mechanism of SKIK through kinetic analysis and to evaluate its applicability to difficult-to-express proteins containing polyproline stretches.
[Method]
For kinetic evaluation, in vitro translation reactions were carried out using mRNAs encoding SKIK-AP-sfGFP. The translation rate was calculated based on the fluorescence intensity of newly synthesized sfGFP monitored in real time. The obtained data were analyzed using the Michaelis–Menten-like model. In addition, SKIK tagging was applied to difficult-to-express proteins, including human interleukin 11 (IL-11), human G protein signaling modulator 3 (GPSM3), and a DUF58 domain–containing protein from Streptomyces sp., all of which contain polyproline residues near the N-terminus. These proteins were synthesized in both an in vitro translation system and an in vivo E. coli expression system, and the products were analyzed by Western blotting.
[Results]
By analyzing the calculated translation-rate data under several assumptions, we found that the data can be plotted and the parameters can be interpreted within a Michaelis–Menten-like model. Furthermore, SKIK tagging tended to increase production yields for proteins containing polyproline, particularly under in vitro translation. In vivo, the extent of the effect varied depending on the properties and types of the target proteins.
[Conclusion]
Effect of translation-enhancing peptide on protein synthesis was kinetically analyzed and evaluated. Kinetic analysis provides quantitative insight into SKIK-mediated translation enhancement, and SKIK tagging is a useful strategy to improve the production of difficult-to-express proteins containing polyproline stretches.
Protein production is crucial for efficient biomanufacturing. However, ribosomal stalling, or translational arrest, induced by polyproline stretches or arrest peptides (APs) is considered one of the bottlenecks in the translation step, often leading to severely reduced yields. Our research group has developed a short translation-enhancing peptide, the SKIK tag (Ser-Lys-Ile-Lys), which promotes translation and increases protein production in Escherichia coli when fused to the N-terminus of target proteins. In addition, SKIK has been shown to alleviate AP-induced translational arrest. However, the detailed mechanism underlying its effect remains unknown. In this study, we aimed to get insights into the translation-enhancing mechanism of SKIK through kinetic analysis and to evaluate its applicability to difficult-to-express proteins containing polyproline stretches.
[Method]
For kinetic evaluation, in vitro translation reactions were carried out using mRNAs encoding SKIK-AP-sfGFP. The translation rate was calculated based on the fluorescence intensity of newly synthesized sfGFP monitored in real time. The obtained data were analyzed using the Michaelis–Menten-like model. In addition, SKIK tagging was applied to difficult-to-express proteins, including human interleukin 11 (IL-11), human G protein signaling modulator 3 (GPSM3), and a DUF58 domain–containing protein from Streptomyces sp., all of which contain polyproline residues near the N-terminus. These proteins were synthesized in both an in vitro translation system and an in vivo E. coli expression system, and the products were analyzed by Western blotting.
[Results]
By analyzing the calculated translation-rate data under several assumptions, we found that the data can be plotted and the parameters can be interpreted within a Michaelis–Menten-like model. Furthermore, SKIK tagging tended to increase production yields for proteins containing polyproline, particularly under in vitro translation. In vivo, the extent of the effect varied depending on the properties and types of the target proteins.
[Conclusion]
Effect of translation-enhancing peptide on protein synthesis was kinetically analyzed and evaluated. Kinetic analysis provides quantitative insight into SKIK-mediated translation enhancement, and SKIK tagging is a useful strategy to improve the production of difficult-to-express proteins containing polyproline stretches.
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