Biomedical subjects

P S Daugherty

Publications and source records attributed to P S Daugherty.

5 recordsLinked to original sources

PubMed2000

Flow cytometric screening of cell-based libraries.

Flow cytometry is a powerful, high-throughput library screening tool in numerous applications including the isolation of bioactive molecules from synthetic combinatorial libraries, the identification of virulence genes in microorganisms, and the study and engineering of protein functions. Using flow cytometry, large libraries of protein mutants expressed in microorganisms can be screened quantitatively for desired functions, including ligand binding, catalysis, expression level, and stability. Rare target cells, occurring at frequencies below 10(-6), can be detected and isolated from heterogeneous library populations using one or more cycles of cell sorting and amplification by growth. Flow cytometry is particularly powerful because it provides the unique opportunity to observe and quantitatively optimize the screening process. However, the ability to isolate cells occurring at such low frequencies within a population requires consideration and optimization of screening parameters. With this aim, an analysis of the various parameters involved in screening cell-based libraries for rare target cells possessing a desired trait is presented.

Cell Separation↗

PubMed2000

Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies.

Random mutagenesis and selection using phage or cell surface display provides an efficient method for affinity maturation of single chain Fv (scFv) antibodies, thereby improving function in various applications. To investigate the effects of mutation frequency on affinity maturation, error-prone PCR was used to generate libraries containing an average (m) of between 1.7 and 22.5 base substitutions per gene in a high affinity scFv antibody that binds to the cardiac glycoside digoxigenin. The scFv antibody libraries were displayed on Escherichia coli, and mutant populations were analyzed by flow cytometry. At low to moderate mutation frequencies with an average mutation rate of m </= 8, the fraction of clones exhibiting binding to a fluorescently labeled conjugate of digoxigenin decreased exponentially (r(2) = 0.99), but the most highly mutated library (m = 22.5) had significantly more active clones than expected relative to this trend. A library with a low error rate (m = 1.7), one with moderate error rate (m = 3.8), and the one with high error rate (m = 22.5) were screened for high affinity clones under conditions of identical stringency using fluorescence-activated cell sorting. After several rounds of enrichment, each of the three libraries yielded clones with improved affinity for the hapten. The moderate and high error rate libraries gave rise to clones exhibiting the greatest affinity improvement. Taken together, our results indicate that (i) functional clones occur at an unexpectedly high frequency in hypermutated libraries, (ii) gain-of-function mutants are well represented in such libraries, and (iii) the majority of the scFv mutations leading to higher affinity correspond to residues distant from the binding site.

Amino Acid Sequence↗

PubMed1999

Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface.

Polypeptide library screening technologies are critically dependent upon the characteristics of the expression system employed. A comparative analysis of the lpp-lac, tet and araBAD promoters was performed to determine the importance of tight regulation and expression level in library screening applications. The surface display of single-chain antibody (scFv) in Escherichia coli as an Lpp-OmpA' fusion was monitored using a fluorescently tagged antigen in conjunction with flow cytometry. In contrast to the lpp-lac promoter, both tet and araBAD promoters could be tightly repressed. Tight regulation was found to be essential for preventing rapid depletion of library clones expressing functional scFv and thus for maintaining the initial library diversity. Induction with subsaturating inducer concentrations yielded mixed populations of uninduced and fully induced cells for both the tet and araBAD expression systems. In contrast, homogeneous expression levels were obtained throughout the population using saturating inducer concentrations and could be adjusted by varying the induction time and plasmid copy number. Under optimal induction conditions for the araBAD system, protein expression did not compromise either cell viability or library diversity. This expression system was used to screen a library of random scFv mutants specific for digoxigenin for clones exhibiting improved hapten dissociation kinetics. Thus, an expression system has been developed which allows library diversity to be preserved and is generally applicable to the screening of E. coli surface displayed libraries.

Antibodies↗

PubMed1998

Antibody affinity maturation using bacterial surface display.

A quantitative system for screening combinatorial single-chain Fv (scFv) antibody libraries was developed utilizing surface display on Escherichia coli and fluorescence-activated cell sorting (FACS). This system was employed to isolate clones with high-affinity to a fluorescently-labeled hapten from libraries constructed by randomizing heavy and light-chain residues in the anti-digoxin 26-10 derived antibody, scFv(dig). The use of flow cytometry enabled the detection of rare library members directly in heterogeneous populations and the optimization of selection conditions prior to sorting. A heavy-chain mutant having wild-type affinity (KD = 0.91+/-0.22 nM) and an expected representation frequency of less than 1 x 10(6), was selected to homogeneity after three rounds utilizing increasingly stringent selection conditions. The isolated clone possessed two distinct point mutations relative to the wild-type DNA sequence, yet still coded for the wild-type amino acid sequence, suggesting that the wild-type residues may be optimal at the randomized positions. An affinity improved clone (KD = 0.30+/-0.05 nM), having a dissociation constant approximately threefold lower than the wild-type antibody, was isolated from a smaller light-chain library in a single sorting step. Flow cytometry was shown to be a simple and rapid method for the determination of the relative hapten dissociation rate constants of selected clones without requiring subcloning. The relative rate constants estimated by FACS were confirmed by producing the scFv antibodies in soluble form and measuring hapten binding kinetics by surface plasmon resonance (SPR). These results demonstrate that E.coli surface display, coupled with quantitative selection and analysis using FACS, has the potential to become a powerful tool for rapid isolation and characterization of desirable mutants from large polypeptide libraries.

Antibody Affinity↗

PubMed1997

Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines.

In recent years there has been considerable progress towards the development of expression systems for the display of heterologous polypeptides and, to a lesser extent, oligosaccharides on the surface of bacteria or yeast. The availability of protein display vectors has in turn provided the impetus for a range of exciting technologies. Polypeptide libraries can be displayed in bacteria and screened by cell sorting techniques, thus simplifying the isolation of proteins with high affinity for ligands. Expression of antigens on the surface of nonvirulent microorganisms is an attractive approach to the development of high-efficacy recombinant live vaccines. Finally, cells displaying protein receptors or antibodies are of use for analytical applications and bioseparations.

Bacterial Proteins↗