TSRI chemists unveil new technique for marking and selecting cells

The technique makes use of the tight binding of two proteins that are cheaply obtainable, but are not found in human or other mammalian cells, giving it distinct advantages over existing cell-marking techniques

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LA JOLLA, Calif.—Seeking an easier and cheaper way to markcells for selectability in molecular biology research, chemists at The ScrippsResearch Institute (TSRI) have designed a new method that allows scientists toadd a marker to certain cells, so that these cells may be easily located and/orselected out from a larger cell population. The technique, described in arecent TSRI study, makes use of the tight binding of two proteins that arecheaply obtainable, but are not found in human or other mammalian cells, givingit distinct advantages over existing cell-marking techniques.
The study, "Engineering Cell Surfaces for OrthogonalSelectability," was published Dec. 13 in the online version of the chemistryjournal Angewandte Chemie InternationalEdition.
The selective addition of markers to expressed proteins hasbecome a standard procedure in molecular biology, with the most frequently usedmarkers being fluorescent proteins and epitope tags. Fluorescent markers suchas green fluorescent protein (GFP), for example, enable the study of thesubcellular localization of the protein by fluorescent microscopy, as well asthe isolation of cells expressing the protein by fluorescent cell sorting.Experimenters have many methods by which to study the expression patterns ofthe marked protein and even isolate the cells expressing it by cell sorting.Importantly, in all these methods, one assumes that the marker does not alterthe properties of the protein to which it is appended. But according to theresearchers, what seems to be missing is a robust, genetic method to mark thesurface of the cells themselves, so that certain cells can be easily and simplyisolated from a population where they may be a minor component. 
Thus, the TSRI team designed a system for the affinityselection of cells and plasma membranes based on the expression of thechitin-binding domain (ChBD) of the enzyme chitinase on the cell surface. Thesystem is "cheap, easy and sensitive," says TSRI Institute Prof. Richard A.Lerner, who is the senior author of the new report.
The system's design is yet another crowning achievement inLerner's long career as a research chemist, as he was the architect of the mostimportant advance since the discovery of monoclonal antibodies a quartercentury ago: the conception, design and creation of combinatorial antibodylibraries, which is currently the most widely used of all libraries in thefield of biochemistry and which enabled a broadening of the scope of action ofthe immune system. Lerner set the stage in an article published in Science in 1989, and all the advancesproduced in the change in combinatorial libraries derived directly orindirectly from this article. In 1991, he identified the essence of theproduction of antibodies without immunization, and his method remains the mostefficient way to produce fully human antibodies. Moreover, Lerner has been apioneer in the development of catalytic antibodies, a strategy to accelerateand catalyze chemical reactions for which traditional methods are notefficient.
Lerner's work has resulted in two top-selling drugs—Abbott'sHumira, a treatment for inflammatory diseases such as rheumatoid arthritis,Crohn's disease and plaque psoriasis; and GlaxoSmithKline's and Human GenomeSciences' Benlysta, a treatment for lupus.
"This research is important in that it further served theantibody libraries that I invented," says Lerner. "We wanted to develop amethod where we could just mix a few cells with the right targets, and a lot ofcells that didn't have the targets, so that when they interact with phage, youcould pick out the right cells and leave the wrong ones behind."
In the basic technique, a new gene can be added to cells withina larger DNA vector that also includes the genetic sequences for ChBD and GFP.Because the ChBD marker, in the vector, is produced in a way that anchors it toa cell's membrane, it also can serve as a powerful tool for selecting just themembrane fraction of a sample of cellular material. The ChBD molecule will beproduced in such a way that it ends up being held on the outer surface of itshost cell's plasma membrane—and the GFP molecule will sit just inside themembrane. The GFP serves as a visual beacon, while the ChBD serves as a handygripping point for cell selection.
After exposing a culture of test cells to this experimentalChBD-containing vector, the scientists were able to see, via the GFP tags, whichcells were expressing them, and were able to select them out easily, with highsensitivity, using magnetic beads coated with chitin. Importantly, theseselected cells could produce progeny cells that seemed normal and healthy.
According to the TSRI team, this method could be importantfor enriching for transformants, especially when the transformation frequencyis low and would facilitate combinatorial antibody selections of phage thatbind to cell surfaces where, as for transformants, the target cell can be aminor component of an otherwise large population. In addition, the method wouldallow for the rapid affinity-based isolation of plasma membranes forbiochemical studies.
"The method should be useful in a variety of applicationsthat require separating out certain types of cells," says Lerner. "If you lookat it from 30,000 feet, it is a cell-service marker, but it's really more thana marker because it slows you to selectively capture the cells that areexpressed."
Lerner and his colleagues are now investigating thepotential use of ChBD-based cell marking in living animals in an attempt totrack the fates of selected cell types throughout an animal's lifespan.
The technology is available to researchers if they contactTSRI's Office of Technology Development, at (858) 784-8140 or through thedepartment's webpage athttp://www.scripps.edu/research/technology/contactus.html.
Other contributors to the study were first author YingjiePeng, a postdoctoral fellow; Teresa M. Jones and Diana I. Ruiz of the TSRIDepartment of Chemistry; and Dae Hee Kim of the Scripps Korea AntibodyInstitute. The work was supported by a grant from the institute.

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