ABSTRACT
Triple-FLAG (3 × FLAG)-tagged proteins can be affinity purified through binding to an anti-FLAG antibody and competitive elution with excess free 3 × FLAG peptide. To expand the availability of the 3 × FLAG purification system, we produced a recombinant His-tagged 3 × FLAG peptide in Brevibacillus choshinensis. The screening of connecting linkers between His-tag and the 3 × FLAG peptide, culture containers, and culture media showed that the His-tagged 3 × FLAG peptide with an LA linker was most expressed in 2SY medium using a baffled shake flask. The peptide was affinity-purified to give a yield of about 25 mg/L of culture. The peptide was effective for eluting 3 × FLAG-tagged α-amylase from anti-FLAG magnetic beads. Finally, the peptide remaining in the amylase fraction was removed by His-tag affinity purification. These results show that the recombinant His-tagged 3 × FLAG peptide can function as an easy-to-remove affinity peptide in the 3 × FLAG purification system.
Subject(s)
Brevibacillus , Recombinant Proteins/metabolism , Brevibacillus/genetics , Brevibacillus/metabolism , Chromatography, Affinity/methods , Peptides/genetics , Peptides/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
We have previously reported that monoclonal antibodies against the (pro)renin receptor [(P)RR] can reduce the Wnt/ß-catenin-dependent development of pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic cancer. Antibodies against two (P)RR regions (residues 47-60 and 200-213) located in the extracellular domain (ECD) reduced the proliferation of human PDAC cells in vitro. Although these regions probably participate in the activation of Wnt/ß-catenin signaling, their functional significance remains unclear. Moreover, the (P)RR ECD is predicted to possess an intrinsically disordered region (IDR), which allows multiple protein interactions because of its conformational flexibility. In this study, we investigated the significance of the two regions and the IDR by in silico 3D structural analysis using the AlphaFold2 program and evolutionary sequence conservation profile. The model showed that ECD adopted a folded domain (residues 17-269) and had an IDR (residues 270-296). The two regions mapped onto the structural model formed a continuous surface patch comprising evolutionarily conserved hydrophobic residues. The homodimeric structure predicted by AlphaFold2 showed that full-length (P)RR comprising the ECD, single-span transmembrane, and cytoplasmic domains formed a twofold symmetric dimer via the ECD, which explains the experimentally proven homodimerization. The dimer model possessed two hand-shaped grooves with residues 47-60 and 200-213 in their palms and the IDR as their fingers. Based on these findings, we propose that the IDR-containing hydrophobic grooves act as a binding site for (P)RR and perform multiple functions, including Wnt signaling activation. Antibodies against the (pro)renin receptor residues 47-60 and 200-213 can inhibit pancreatic ductal adenocarcinoma (PDAC) cell proliferation by suppressing Wnt signaling. This study provides 3D structural insights into receptor binding and one-to-many interactions, which underpin the functional versatility of this receptor.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , beta Catenin/metabolism , Binding Sites , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Prorenin Receptor , Protein Binding , Pancreatic NeoplasmsABSTRACT
Carnosine is a naturally occurring dipeptide and a functional component in foods, while also showing health-promoting effects. Generally, food-derived carnosine is quantified via high-performance liquid chromatography (HPLC). We have developed a method for quantifying carnosine in foods using microbial enzymes, ß-Ala-Xaa dipeptidase (BapA) and histidine ammonia-lyase (HAL). The carnosine concentrations in extracts of chicken, pork, beef, bonito, and tuna were determined via both HPLC and enzymatic determination. The carnosine contents measured via enzymatic determination were in agreement with those determined via conventional HPLC analysis. Relative standard-deviation values of the conventional HPLC method and the enzymatic determination of carnosine in foods were 0.728-5.76% and 0.504-4.58%, respectively. The recovery of carnosine in food extracts via enzymatic determination was 97-103%. Therefore, the developed enzymatic determination technique using BapA and HAL can be used for the determination of carnosine in meats and fishes with comparable accuracy to that of conventional HPLC analysis.
