Abstract
Protein engineering—the process of developing useful or valuable proteins—has successfully created a wide range of proteins tailored to specific agricultural, industrial, and biomedical applications. Protein engineering may rely on rational techniques informed by structural models, phylogenic information, or computational methods or it may rely upon random techniques such as chemical mutation, DNA shuffling, error prone polymerase chain reaction (PCR), etc. The increasing capabilities of rational protein design coupled to the rapid production of large variant libraries have seriously challenged the capacity of traditional screening and selection techniques. Similarly, random approaches based on directed evolution, which relies on the Darwinian principles of mutation and selection to steer proteins toward desired traits, also requires the screening of very large libraries of mutants to be truly effective. For either rational or random approaches, the highest possible screening throughput facilitates efficient protein engineering strategies. In the last decade, high-throughput screening (HTS) for protein engineering has been leveraging the emerging technologies of droplet microfluidics. Droplet microfluidics, featuring controlled formation and manipulation of nano- to femtoliter droplets of one fluid phase in another, has presented a new paradigm for screening, providing increased throughput, reduced reagent volume, and scalability. We review here the recent droplet microfluidics-based HTS systems developed for protein engineering, particularly directed evolution. The current review can also serve as a tutorial guide for protein engineers and molecular biologists who need a droplet microfluidics-based HTS system for their specific applications but may not have prior knowledge about microfluidics. In the end, several challenges and opportunities are identified to motivate the continued innovation of microfluidics with implications for protein engineering.
Highlights
Engineered proteins with tailored properties evolved from natural precursors have been playing an increasingly important role in a spectrum of agricultural, industrial, and biomedical applications [1,2]
Often, when a cell based fluorescent readout is achievable, screening of engineered protein libraries is performed with fluorescence-activated cell sorting (FACS) to separate a population of cells into sub-populations based on fluorescent labeling [22]
The results showed that an enrichment of 30,000-fold was achieved for a 1:100,000 dilution (0.001%) of Green Fluorescent Protein (GFP) expressing versus non-GFP expressing cells, which is comparable to enrichment rates previously reported [21,29,68]
Summary
Engineered proteins with tailored properties evolved from natural precursors have been playing an increasingly important role in a spectrum of agricultural, industrial, and biomedical applications [1,2]. Often, when a cell based fluorescent readout is achievable, screening of engineered protein libraries is performed with fluorescence-activated cell sorting (FACS) to separate a population of cells into sub-populations based on fluorescent labeling [22] In this case, the phenotype-genotype connection is unbroken because the cells are selected directly. Miniaturization of screening systems can substantially increase sorting efficiency, improve selection and reduce screening costs, enabling exploration of very large libraries (108–109). These advantageous properties have stimulated emerging micro- and nanotechnologies to move into applications in Micromachines 2019, 10, x. Arolptelrent astoivrteilnyg, th(FeAcoDnSt)e.nAt oltfedrnroaptilveetslyo,ftihnetecreosnttceannt boef drercoopvlertsedofviinatflerueosrtecsacnenbceer-aectoivearted evlieacftlruoocoreaslecescnecnec-aec(tFivAaEte).d electrocoalescence (FAE)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
