Ribosomal stalling is a phenomenon that occurs during the translation of mRNA into proteins when the ribosome encounters a specific sequence or structural element that temporarily halts its progression along the mRNA strand. This process can have important implications for the production of recombinant therapeutic proteins, which are proteins engineered and produced in host cells for use as medical treatments. In the context of recombinant protein production, ribosomal stalling can be problematic because it can lead to reduced protein yields and lower production efficiency. When the ribosome stalls at a specific mRNA sequence or structure, it can lead to a delay in the overall translation process. This delay can reduce the rate at which the recombinant protein is synthesized, resulting in lower protein yields over a given time frame. This is particularly concerning in biomanufacturing, where maximizing protein production is essential to meet the demand for therapeutic proteins. Ribosomal stalling can also lead to the premature termination of translation or the synthesis of incomplete protein fragments. This can result in the production of non-functional or misfolded proteins that are either degraded by the cellular quality control machinery or are inactive when used as therapeutic agents. Such aberrant proteins are not only a waste of resources but can also pose safety risks to patients. Moreover, prolonged ribosomal stalling can trigger cellular stress responses, including the unfolded protein response (UPR). The UPR aims to restore cellular homeostasis by reducing the overall protein synthesis rate and increasing the production of chaperone proteins to assist with protein folding. These cellular responses can divert cellular resources away from recombinant protein production and further reduce yields. To mitigate the negative effects of ribosomal stalling on recombinant protein production, several strategies can be employed including reengineering the coding sequence of the recombinant protein to minimize the presence of rare codons or sequences prone to ribosomal stalling can improve translation efficiency. Codon optimization involves replacing codons that are known to cause ribosomal stalling with synonymous codons that are translated more efficiently by the ribosome.
In a new study published in the Journal of Biological Chemistry and led by Dr. Teruyo Ojima-Kato, Dr. Yuma Nishikawa, Dr. Yuki Furukawa, Dr. Takaaki Kojima, and Dr. Hideo Nakano from the Nagoya University addressed a persistent challenge in biotechnology: the low yield of protein production in microbial hosts, a hurdle in various applications including therapeutic protein production. This is a critical issue in E. coli, a widely used host for recombinant protein production. The study’s focus on the MSKIK peptide emerges from the need to understand and mitigate ribosomal stalling, particularly in the context of arrest peptides like SecM and CmlA leader peptide.
The authors introduced a novel approach by incorporating the MSKIK peptide sequence into the E. coli system. This peptide, previously understudied, was found to increase the production of difficult-to-express proteins when inserted into the N-terminus of the genes.. The researchers employed a series of in vivo and in vitro experiments, showcasing the versatility and robustness of the MSKIK peptide in different settings and conditions. They demonstrated that the MSKIK peptide effectively prevents or resolves ribosomal stalling, leading to enhanced protein production. This is particularly noteworthy in the context of SecM and CmlA arrest peptides. An interesting aspect of the research is the examination of SKIK-encoding codons. The authors confirmed that the benefits of the MSKIK peptide are not due to codon optimization but are inherent to the peptide sequence itself.
The primary implication of Teruyo Ojima-Kato and colleagues’ study is the potential for increased yield in protein production. This has far-reaching consequences in both research and industrial applications, particularly in the manufacturing of therapeutic proteins. The ability to enhance protein production efficiently can revolutionize the production of therapeutic proteins, making them more accessible and reducing costs. Moreover, the methodologies employed in the study contribute to a deeper understanding of the protein synthesis process, providing a template for future research in this area.
While the team highlighted the effectiveness of the MSKIK peptide, the exact mechanism of its action remains to be fully elucidated. Understanding this mechanism is crucial for applying this knowledge to other systems and potentially to other organisms. Investigating the application of the MSKIK peptide in other expression systems or organisms could expand its utility and impact. It will be noteworthy exploring the potential of the MSKIK peptide in clinical settings, particularly in the production of therapeutic proteins, is a promising area for future research. In conclusion, the study on the nascent MSKIK peptide presents a significant breakthrough in the field of protein production, addressing a longstanding challenge in biotechnology and the benefits extend beyond E. coli, offering potential advancements in biotechnology and medicine.
Ojima-Kato T, Nishikawa Y, Furukawa Y, Kojima T, Nakano H. Nascent MSKIK peptide cancels ribosomal stalling by arrest peptides in Escherichia coli. J Biol Chem. 2023 ;299(5):104676. doi: 10.1016/j.jbc.2023.104676.