High-throughput screening of enzymes by retroviral display using droplet-based microfluidics.

During the last 25 years, display techniques such as phage display have become very powerful tools for protein engineering, especially for the selection of monoclonal antibodies. However, while this method is extremely efficient for affinity-based selections, its use for the selection and directed evolution of enzymes is still very restricted. Furthermore, phage display is not suited for the engineering of mammalian proteins that require posttranslational modifications such as glycosylation or membrane anchoring. To circumvent these limitations, we have developed a system in which structurally complex mammalian enzymes are displayed on the surface of retroviruses and encapsulated into droplets of a water-in-oil emulsion. These droplets are made and manipulated using microfluidic devices and each droplet serves as an independent reaction vessel. Compartmentalization of single retroviral particles in droplets allows efficient coupling of genotype and phenotype. Using tissue plasminogen activator (tPA) as a model enzyme, we show that, by monitoring the enzymatic reaction in each droplet (by fluorescence), quantitative measurement of tPA activity in the presence of different concentrations of the endogenous inhibitor PAI-1 can be made on-chip. On-chip fluorescence-activated droplet sorting allowed the processing of 500 samples per second and the specific collection of retroviruses displaying active wild-type tPA from a model library with a 1000-fold excess of retroviruses displaying a non-active control enzyme. During a single selection cycle, a more than 1300-fold enrichment of the active wild-type enzyme was demonstrated.

A competition-based assay for the screening of species-specific antibiotics.

OBJECTIVES: To develop a high throughput screening-compatible assay for the selection of species-specific antibiotics that do not harm human cells. METHODS: Staphylococcus aureus and human reporter cells continuously generating a fluorescence signal were competitively co-cultivated. The fluorescence signals were determined in the presence and absence of the specific antibiotic streptomycin and the toxic compound sodium azide. The results were compared with a standard cfu assay. RESULTS: In the absence of an effective antibiotic, S. aureus outgrew the human reporter cells and thus abolished the fluorescence signal. Conversely, the addition of streptomycin resulted in the growth of the reporter cells and a strong fluorescence signal. When sodium azide was added instead of streptomycin, only a very low background signal was obtained indicating toxicity and damage to the human reporter cells. The assay proved to be highly reliable (Z-factor >0.9) and high fluorescence signals correctly correlated with the efficient inhibition of S. aureus, as determined in comparative cfu assays. CONCLUSIONS: In contrast to conventional cfu assays, the co-cultivation system allows the effects of a drug candidate on pathogens and human cells to be monitored simultaneously. Cytotoxic compounds can, therefore, be quickly ruled out during a primary screen. The nature of the screen also enables effective antibiotics to be identified without engineering the target pathogen to yield a fluorescence signal.