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261.
262.
Although various methods for selective protein tagging have been established, their ap plications are limited by the low fluorescent tagging efficiency of specific terminal regions of the native proteins of interest (NPIs). In this study, the highly sensitive fluorescence imaging of single NPIs was demonstrated using a eukaryotic translation mechanism involving a free carboxyl group of a cell-permeable fluorescent dye. In living cells, the carboxyl group of cell-permeable fluorescent dyes reacted with the lysine residues of acceptor peptides (AP or AVI-Tag). Genetically encoded recognition demonstrated that the efficiency of fluorescence labeling was nearly 100%. Nickel-nitrilotriacetic acid (Ni-NTA) beads bound efficiently to a single NPI for detection in a cell without purification. Our labeling approach satisfied the necessary conditions for measuring fluorescently labeled NPI using universal carboxyl fluorescent dyes. This approach is expected to be useful for resolving complex biological/ecological issues and robust single-molecule analyses of dynamic processes, in addition to applications in ultra-sensitive NPIs detection using nanotechnology. 相似文献
263.
构建含SUMO、IF2、GST、NusA、MsyB、Trx和MBP融合标签的重组表达载体,转化到大肠杆菌E.coli Transetta (DE3)中进行自诱导(auto-induction,AI)表达,以提高T4 DNA连接酶(T4 DL)的表达产量。通过磁珠法检测融合蛋白的可溶性表达情况,10% SDS-PAGE电泳结果显示,重组菌Transetta (DE3)(pNBEVⅡ-T4 DL)诱导表达的可溶性融合蛋白Trx-T4 DL的产量最多,经诱导培养条件优化后,Trx-T4 DL的可溶性大幅度提高,确定了最佳诱导条件为30℃、装瓶量50 mL/250 mL、接种量2‰、pH7。分别用镍柱和MagNi磁珠纯化重组菌破碎后上清中的融合蛋白Trx-T4 DL,结果显示后者纯化效率更高,最终获得的融合蛋白浓度为1700.462 mg/L。与其他公司T4 DL活性进行比较,检测其酶活性约为500 U/μL,并使其成功应用于低背景重组克隆载体构建中,为融合蛋白Trx-T4 DL的生产及应用提供理论基础。 相似文献
264.
Alexander J. Lander Dr. Yi Jin Dr. Louis Y. P. Luk 《Chembiochem : a European journal of chemical biology》2023,24(4):e202200537
Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery of non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities. 相似文献
265.
Yuen Yi Lam Dr. Angel Tan Cameron J. Nowell Dr. Kristian Kempe Prof. Ben J. Boyd 《Chembiochem : a European journal of chemical biology》2023,24(12):e202300144
Metabolic oligosaccharide engineering (MOE) of cells with synthetic monosaccharides can introduce functionality to the glycans of cell membranes. Unnatural sugars (e. g., peracetylated mannose-azide) can be expressed on the cell surface with the azide group in place. After MOE, the azide group can participate in a copper-free click reaction with an alkyne (e. g., dibenzocyclooctyne, DBCO) probe. This allows the metabolic fate of monosaccharides in cells to be understood. However, in a drug delivery context it is desirable to have azide groups on the probe (e. g. a drug delivery particle) and the alkyne (e. g. DBCO) on the cell surface. Consequently, the labelling efficiency of intestinal cell lines (Caco-2 and HT29-MTX-E12) treated with N-dibenzocyclooctyne-tetra-acetylmannosamine, and the concentration- and time-dependent labelling were determined. Furthermore, the labelling of mucus in HT29-MTX-E12 cells with DBCO was shown. This study highlights the potential for using MOE to target azide-functionalised probes to intestinal tissues for drug delivery applications. 相似文献
266.
Miao Liu Yuhan Wang Zhicheng Yan Jiabin Yang Prof. Yongyou Wu Prof. Dawei Ding Prof. Dr. Xingyue Ji 《Chembiochem : a European journal of chemical biology》2023,24(3):e202200506
Bioorthogonal prodrugs with both fast reaction kinetics and multiple outputs are highly desirable but are only found sporadically. Herein, we report a novel photoclick-and-release strategy for the co-activation of carbon monoxide and a self-reporter, carbonyl sulfide, or sulfonamide with fast reaction kinetics (k: 1.4–22.6 M−1 s−1). Such a photoclick-and-release strategy was successfully applied in live cells to deliver carbon monoxide and a fluorescent self-reporter, both of which exhibited pronounced antiproliferative activity against 4T1 cancer cells. It is conceivable that this photoclick-and-release strategy could find applications in other fields, in which a controlled bond cleavage is preferred. 相似文献
267.
Seungnyeong Heo Jonghwi Baek Juhyun Bae Prof. Dr. Bo Am Seo Seongjun Kim Subin Jeong Suhyun Kim Dr. Yiseul Ryu Prof. Dr. Joong-jae Lee 《Chembiochem : a European journal of chemical biology》2023,24(11):e202200700
Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost-effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag-SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co-expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high-yield production of recombinant HRP variants and other difficult-to-express proteins in bacteria without complex downstream processes. 相似文献
268.
Tyrza van Leeuwen Dr. Ward Doelman Robin W. R. van den Kieboom Dr. Bogdan I. Florea Prof. Sander I. van Kasteren 《Chembiochem : a European journal of chemical biology》2023,24(8):e202300082
Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass-spectrometry based proteomics – the ideal analysis tool in this case – requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click-antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG-based Rink amide resin, that enables capture of click-antigens via copper-catalyzed azide-alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a-specific background material, prior to retrieval peptides by acid-mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha-labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures. 相似文献
269.
Dr. Han Zhou Tomaya Carpenter Xuan Fu Bo Jin Britton Ody Mohammad Sazid Hassan Savannah E. Jacobs Jenny Cheung Eve M. Nicholson Prof. Dr. Mark Turlington Prof. Dr. Bo Zhao Dr. Sonja Lorenz Prof. Dr. T. Ashton Cropp Prof. Dr. Jun Yin 《Chembiochem : a European journal of chemical biology》2022,23(8):e202200133
Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) Nϵ-L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination. 相似文献
270.
Zhentao Shao Dr. Yingdi Zhu Prof. Juan Li 《Chembiochem : a European journal of chemical biology》2023,24(9):e202200763
Non-enzymatic post-translational modifications (nPTMs) have been proposed as indicators of cellular stresses and diseases. Unfortunately, direct assessment of nPTMs in native environment is extremely challenging due to the heterogeneity of adducts and the lack of tagging tools. Given these challenges, bioorthogonal probes (BPs) have been developed for the analysis of nPTMs. The rationality is that BPs could selectively install azides or alkynes into nPTMs as a biorthogonal handle for the following enrichment or tracking. Herein, we review the state-of-art of BPs used for nPTMs studies, clarify their working principles, and highlight how they advance our understanding of the biological functions of nPTMs. 相似文献