Cells [28] has been employed successfully in cell sorting for improved affinity
Cells [28] has been employed successfully in cell sorting for improved affinity of single chain Fv antibody fragments out of mutant libraries. As an extreme, a dissociation constant Kd 50 fM was achieved after four rounds of mutagenesis with error prone PCR and FACS screening of 105?106 cells each [29].Screening for enzymatic activity Screening for ligand binding is quite straight forward in a cell sorting frame, as binding of the fluorescent probe to the cell is an intrinsic feature of the desired setup. On the contrary, a system for the screening of novel or improved enzymatic activities has to be much more elaborate. The major problem to solve is to link the signal (a product of the desired reaction) to the cell that produced an enzyme catalyzing this reaction. While it is rather straightforward to link an enzyme (or a mutated library) to the expressing cell via one of the above described cell surface display systems, the linkage of enzymatic reaction products is more limited and needs consideration in every case. Olsen et al. have employed the negative charge of the cell surface to bind a protease substrate containing a positively charged moiety. The generation of a fluorescence signal upon proteolytic cleavage was enabled by a fluorescence resonance energy transfer (FRET) quenching pair of dyes that was separated upon cleavage, leaving only the fluorescent partner containing25 the positive charges on the cell surface. Using the surface bound serine protease OmpT as a model, a 5000 fold enrichment of E. coli expressing active OmpT out of cells expressing an inactive variant PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 could be achieved, as well as the isolation of OmpT variants with an altered substrate specificity with 60 fold enhanced catalytic activity [35]. Recently, this group has demonstrated the simultaneous screening for activity and selectivity by FACS, using again OmpT as a model [36].Apart from selecting antibody mutants, surface display and FACS have also been applied for the screening of peptide libraries for ligand binding peptides. Escherichia coli surface display was employed in a combined MACS and FACS procedure to isolate peptides that bind with high affinity to different protein ligands [20]. Alternatively, Wernerus and Stahl [21] described the development of a stable Staphylococcus carnosus surface display system, its usability for FACS screening, and its application for the screening of ligand binding peptides based on Staphylococcus aureus protein A domains, so-called affibody ligands [30]. Viewing beyond the microbial world, baculovirus was exploited as a carrier for surface displayed proteins [31,32]. Peptides or even complex proteins can be displayed either on the virion [31] or on the infected insect cell [33]. Consequently, the selection of displayed peptide sequences was achieved by FACS screening of the Sf9 cells infected with a baculovirus surface display library [34].An example of intracellular enzyme evolution with the aid of FACS was described by Santoro et al. [37]. Using a cascade of T7 RNA polymerase containing amber stop codons, and green fluorescent protein (GFP), they Setmelanotide chemical information screened for mutants of aminoacyl-tRNA synthetase finally leading to the incorporation of unnatural amino acids into proteins. Kawarasaki and coworkers [38] described the screening of intracellular activity of glutathione S-transferase using a fluorogenic product that was contained within the cells. A more radical approach to screening for optimized enzymes is based on cell.
