LABELING OF PROTEIN WITH A NEW EUROPIUM CHELATE OF 5-CHLOROSULFOYL-2-THENOYLTRIFLUOROACETONE(CTTA)AND APPLICATION TO IMMUNOASSAY

We describe for the first time the synthesis and the optimal conditions for protein labeling with a new fluorescent probe,5-chlorosulfoyl-2-thenoyltrifluoroacetone(CTTA),whicb forms a highly fluorescent conplex with Eu^(3+) when conjugated to protein.The labeled proteins were characterized by absorbance and fluorescence measurements and the effect of labeling on the biological activity of sone proteins was also studied.It is shown that the new label is suitable for applications in time-resolved fluoroimmunoassay.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

Electron-withdrawing inductive effects enhanced strategy for protein thiol sensing and blocking agent design

It is a great challenge to discover novel chemical reactions suitable for biological analysis in a living system.The development of novel protein thiol blocking agents is a crucial need for exploring protein thiol functions in protein refolding,signal transduction,and redox regulation.We are always keen on seeking novel chemical reactions applied to endogenous biological macromolecules or protein thiol sensing,blocking,and labeling.In the present work,we have successfully developed a novel agent to block protein thiol by enhanced electron-withdrawing inductive effects.This sensing and blocking process was detailedly monitored by UV-vis,fluorescent spectra,and SDS-Page gel separation.The spectral studies demonstrated that the agent could react ultrafastly with thiol within seconds atμM level.Furthermore,fluorescent imaging in cells and in vivo was further used for the validation of its ability to sensing and blocking thiol,providing evidence of downregulated protein thiols in Parkinson's disease.The enhanced electronwithdrawing inductive effect strategy in this work may provide a general guideline for designing protein thiol agent.

Chemical tags and beyond:Live-cell protein labeling technologies for modern optical imaging

Imaging proteins with high resolution is crucial for studying cellular physiology and pathology.Fluorescence imaging is a privileged method to visualize proteins with subcellular precision in live cells.In recent years,there has been a tremendous advance in the field of fluorescent dyes that are optically more sophisticated than genetically-encodable fluorescent proteins.In this review,we aim to discuss modern bioconjugation methods to specifically incorporate these dyes into protein-of-interests.We focus on advances in live-cell labeling strategies and fluorescent probes,especially the HaloTag,SNAP-tag,TMP-tag,and unnatural amino acid systems and their applications.These protein labeling methods,along with cutting-edge dyes and novel microscopy methods,have become the infrastructure for biological research in the era of super-resolution imaging.

A versatile AIE fluorogen with selective reactivity to primary amines for monitoring amination,protein labeling,and mitochondrial staining

Specific bioconjugation for native primary amines is highly valuable for both chemistry and biomedical research.Despite all the efforts,scientists lack a proper strategy to achieve high selectivity for primary amines,not to mention the requirement of fast response in real applications.Herein,we report a chromone-based aggregation-induced emission(AIE)fluorogen called CMVMN as a self-reporting bioconjugation reagent for selective primary amine identification,and its applications for monitoring bioprocesses of amination and protein labeling.CMVMN is AIE-active and capable of solid-state sensing.Thus,its electrospun films are manufactured for visualization of amine diffusion and leakage process.CMVMN also shows good biocompatibility and potential mitochondria-staining ability,which provides new insight for organelle-staining probe design.Combined with its facile synthesis and good reversibility,CMVMN would not only show wide potential applications in biology,but also offer new possibilities for molecular engineering.

SNAP-tag fluorogenic probes for wash free protein labeling

Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusion tags has been widely used and already commercially available. Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported. Owing to turn-on fluorescence response, fluorogenic probes for SNAP-tag minimize the fluorescence background caused by unreacted or nonspecifically bound probes and allow for direct imaging in living cells without wash-out steps. Thus,real-time analysis of protein localization, dynamics and interactions has been made possible by SNAP-tag fluorogenic probes. In this review,we describe the design strategies of fluorogenic probes for SNAP-tag and their applications in cellular protein labeling.

A mini-review on the enzyme-mediated manipulation of proteins/peptides

Recent years have seen an ever increasing number of enzyme mediated protein/peptide modification reactions, which contribute significantly to the elucidation of related biological functions. The many available enzymes have, however, caused difficulties for practitioners in choosing the most appropriate enzyme for a certain purpose. This review surveyed the widely used enzymes(i.e., sortases, butelase 1,subtiligase, formylglycine generating enzyme and farnesyltransferase) in the manipulation of proteins/peptides, and the application fields of these enzymes as well as the advantages and limitations of each enzyme are summarized.

Emerging markers for antimicrobial resistance monitoring

The appearance and spread of antibiotic-resistant pathogens known as antimicrobial resistance(AMR)is one of the major worldwide health crises that humanity have to deal with over the next decades.One of the main methods for addressing AMR is the effective screening for antimicrobial insensitivity in clinical and environmental monitoring.Current clinical laboratory procedures use traditional culturebased antibiotic susceptibility testing(AST)methods,which can take up to 24 h to identify which drug is suitable for the infection inhibition.Therefore,it is vital to develop novel strategies that offer quick,simple,affordable,reliable,sensitive and accurate AMR monitoring.Sensors for AMR markers detection could possess the essential qualities for quickly identifying resistant microorganisms and could give vital data for the selection of antibacterial drugs administration.This review offers a summary of the innovative application of these AMR markers detection strategies focusing on healthcare and environmental surveillance for the AMR genotypic or phenotypic assessment.