Biology subjects

Ojima-Kato, T.

Publications and source records attributed to Ojima-Kato, T..

6 recordsLinked to original sources

bioRxiv2025

Screening and machine-learning assisted prediction of translation-enhancing peptides reducing ribosomal stalling in Escherichia coli

We previously reported that the nascent SKIK peptide enhances translation and alleviates ribosomal stalling caused by arrest peptides (APs) such as SecM and polyproline when positioned immediately upstream of the APs in both Escherichia coli in vivo and in vitro translation systems. In this study, we performed a comprehensive screening of translation-enhancing peptides (TEPs) using a randomized artificial tetrapeptide library. The screening was based on the ability of the peptides to suppress SecM AP-induced translational stalling in E. coli cells. Various TEPs exhibiting a range of translation-enhancing activities were identified. In vitro translation analysis suggested that the fourth amino acid in the tetrapeptide plays a key role in reducing SecM AP-mediated stalling. Furthermore, we developed a machine learning model using a random forest algorithm to predict TEP activity. The predicted values showed a strong correlation with experimentally measured activities. These findings offer a compact peptide toolkit and a data-driven approach for mitigating AP-induced ribosome stalling, with potential applications in synthetic biology.

synthetic biology↗

bioRxiv2024

Improvement of Antibody Affinity and Using PURE Ribosome Display and Microbial Secretion System

A method has been developed for efficiently enriching and analyzing high-affinity antibody variants by combining the PURE ribosome display (ribosome display using purified cell-free protein synthesis components) with next-generation sequencing (NGS) and the Brevibacillus choshinensis secretion system, using the NZ-1 antibody, which targets the PA tag peptide (GVAMPGAEDDVV), as a model antibody. An artificial DNA encoding the single-chain fragment of binding (scFab) of the NZ-1 antibody was synthesized and actively expressed by cell-free protein synthesis system (CFPS). Region-specific saturation mutations were introduced into the scFab gene based on its structural information. The resulting scFab library was selected against the PA tag through two rounds of PURE ribosome display, followed by Illumina sequencing to identify potential scFab variants with higher affinity. The candidates were expressed as Fab fragments using the B. choshinensis secretion system. These Fab fragments were then purified from the culture supernatant using two-step column chromatography. The binding affinity of the purified Fab was evaluated using a biolayer interferometry assay, revealing a variant Fab with higher affinity than the wild-type Fab. These results demonstrate that integrating PURE ribosome display with NGS analysis and the B. choshinensis secretion expression system enables the rapid identification and analysis of high-affinity antibody variants.

synthetic biology↗

bioRxiv2024

Investigation of Intestinal Bacteria-binding Monoclonal Antibodies Derived from Rabbit Single B Cells

Immunoglobulins in the intestine have been reported to play a pivotal role in regulating the composition and function of intestinal microbiota. However, the relationship between monoclonal antibodies (mAbs) and intestinal bacteria remains elusive due to the lack of analytical methods. Here we report the utilization of Ecobody technology, a single B cell technique developed by our group, for obtaining and investigating intestinal bacteria-binding mAbs derived from single B cells of a non-immunized rabbit. Bacteroides cellulosilyticus isolated from the rabbit stool was used as the antigen for selecting B cells, followed by antibody gene amplification, mAb expression in cell-free protein synthesis (CFPS), and activity evaluation. For large-scale and cost-effective production of the selected mAb, we employed the secretion expression system for antigen-binding fragment (Fab) using Brevibacillus choshinensis, and successfully yielded [~]2.0 mg/L-culture of purified antibody with high affinity to the target bacterium. Purified Fab showed binding activity towards various intestinal bacteria, suggesting the broad specificity toward intestinal bacteria-associated antigens.

bioengineering↗

bioRxiv2024

Effect of translation enhancing nascent SKIK peptide on the arrest peptides containing consecutive Proline

Ribosome arrest peptides (RAPs) such as SecM arrest peptide (SecM AP) 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 consisted of four amino acid residues Ser-Lys-Ile-Lys, effectively alleviate 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) which alleviate ribosome stalling in consecutive-Pro rich protein, synergistically promote translation. A kinetic analysis based on the generation of super folder 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/582505v2_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@c97150org.highwire.dtl.DTLVardef@12e3fb1org.highwire.dtl.DTLVardef@157af4forg.highwire.dtl.DTLVardef@1b67da9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Rapid and cost-effective epitope mapping using PURE ribosome display coupled with next-generation sequencing and bioinformatics

A novel, efficient and cost-effective approach for epitope identification of an antibody has been developed using a ribosome display platform. This platform, known as PURE ribosome display, utilizes an Escherichia coli-based reconstituted cell-free protein synthesis system (PURE system). It stabilizes the mRNA-ribosome-peptide complex via a ribosome-arrest peptide sequence. This system was complemented by next-generation sequencing (NGS) and an algorithm for analyzing binding epitopes. To showcase the effectiveness of this method, selection conditions were refined using the anti-PA tag monoclonal antibody with the PA tag peptide as a model. Subsequently, a random peptide library was constructed using 10 NNK triplet oligonucleotides via the PURE ribosome display. The resulting random peptide library-ribosome-mRNA complex was selected using a commercially available anti-HA (YPYDVPDYA) tag monoclonal antibody, followed by NGS and bioinformatic analysis. Our approach successfully identified the "DVPDY" sequence as an epitope within the hemagglutinin amino acid sequence, which was then experimentally validated. This platform provided a valuable tool for investigating continuous epitopes in antibodies.

bioengineering↗

bioRxiv2022

Nascent MSKIK peptide prevents or releases translation arrest in Escherichia coli

The insertion of the sequence encoding SKIK peptide adjacent to the M start codon of a difficult-to-express protein enhances protein production in Escherichia coli. In this report, we show that the increased production of the SKIK-tagged protein is not due to codon usage of the SKIK sequence. In addition, insertion of SKX, KKX, and AKX (X = G, L, H, Y, E, and F) at the N-terminus increased protein production. Furthermore, insertion of MSKIK just before the SecM arrest peptide (FSTPVWISQAQGIRAGP), which causes translational stalling on mRNA, greatly increased the production of the protein containing the SecM arrest peptide in the E. coli reconstituted cell-free protein synthesis system (PURE system). A similar phenomenon was observed for CmlA leader whose arrest is induced by chloramphenicol. These results suggest that the nascent MSKIK peptide prevents or releases translational stalling immediately following its generation during the translation process.

biochemistry↗