Tracking Endocytosis And Transport Of Mammalian Cell-displayed Proteins With Quantum Dots

Mammalian cell display technology uses recombinant DNA technology to express and localize exogenous peptides or proteins on the cell surface.In contrast to phage display technology and bacterial surface display technology,mammalian cell display technology can guide protein folding and provide various post-translational processing functions,resulting in expressed products that are closest to natural biological protein molecules in terms of molecular structure,physicochemical properties,and biological function.This technology has been successfully applied to directed evolution of enzymes,peptide library screening,high-affinity antibody sorting,antigen/antibody library construction,and new applications continue to emerge.Current studies on mammalian cell display technology are focused on the function of the displayed proteins,while less attention has been paid to the "fate" of these proteins after they are displayed on the cell membrane.The fundamental questions such as how display proteins re-enter the cell and their intracellular transport are still unclear,which greatly limits the widespread use of display technology.Therefore,it is necessary to investigate the "fate" of display proteins.However,traditional research methods usually analyze fixed cells to obtain static,overall averaged results,and are not allowing for real-time,dynamic studies the mechanisms of displayed protein endocytosis and transport in living cells.Single-particle tracking is a microscopy technique for visualizing the trajectory of individual particles or individual molecules.The technique allows the acquisition of information related to particle motion from time-series microscopy images and has become a powerful tool for studying the dynamic processes of biological events within living cells.The prerequisite for implementing single particle tracing is the fluorescent labeling of the individual biomolecules being traced,resulting in real-time,in situ,and prolonged tracing of them.Quantum dots(QDs)have unique optical properties and therefore offer significant advantages in fluorescence imaging.Their high fluorescence intensity improves detection sensitivity and enables the acquisition of high contrast images.In addition,the excellent photostability of QDs enables continuous and longterm tracing of individual target molecules at high temporal resolution.However,QDs do not have recognition capabilities and require functionalization of quantum dots to achieve specific fluorescent labeling of target molecules.In this paper,the endocytosis and transport of the displayed proteins in living cells were studied in real-time and in situ,using the tag protein Halo Tag as a model protein for mammalian cell display technology,combined with quantum dot-based single particle tracer technology.The specific work is as follows:(1)CdZnSeS quantum dots with high fluorescence quantum yield were first synthesized in the organic phase by thermal injection,and then successfully obtained water-dispersible quantum dots by hydrophobic coating with octylamine-grafted polyacrylic acid(OPA),and further coupled with polyethylene glycol to reduce the nonspecific adsorption between quantum dots and cells to obtain the best bioimaging performance.(2)The tag protein Halo Tag is inserted into the expression vector p Display used for the display technology and displayed it on the cell surface as a model protein for the mammalian cell display technology.Halo Tag protein can bind specifically to the haloalkane derivative ligand(Halo Tag ligand)by covalent bonding,and the displayed Halo Tag can be specifically fluorescently labeled using quantum dots modified with Halo Tag ligands.With the help of QDs labeling it was found that the displayed Halo Tag is not always anchored to the cell membrane,but is internalized into the cell in a time and temperature-dependent manner.The endocytosis mechanism of Halo Tag was further investigated by co-localization experiments and drug inhibition assays,and it was found that the displayed protein could enter the cell through clathrin-mediated endocytosis and move along microfilaments and microtubules to lysosomes around the nucleus.It was also observed that some of the endocytosed display proteins were recycled from the near-nuclear region of the cell to the cell surface by circulating endosomes,suggesting that the total amount of display proteins on the cell membrane surface may be related to intracellular dynamic processes such as internalization and recycling.The above results provide support for studying the intracellular dynamic behavior of displayed proteins and are expected to facilitate the optimization and development of mammalian cell display technology.