ABSTRACT
Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR's specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.
Subject(s)
Enzyme Assays/methods , HIV Protease/metabolism , HIV-1/enzymology , Interferometry/methods , Biosensing Techniques , Cloning, Molecular , HIV Protease/genetics , HIV Protease/isolation & purification , Humans , Kinetics , Proteolysis , Recombinant Proteins , Sequence Analysis, DNA , Substrate SpecificityABSTRACT
In response to societal grand challenges, professors have unique opportunities to effect change, repurposing their expertise to deploy relevant, timely, practical, and research-backed knowledge for the betterment of communities. Drawing on scholarship on postcrisis organizing, entrepreneurial hustle, and social entrepreneurship, we provide a firsthand, real-time case description of a three-day "virtual idea blitz" organized in response to the COVID-19 crisis. The event was organized and executed in less than a week and ultimately involved 200 individuals, including entrepreneurs, coders, medical doctors, venture capitalists, industry professionals, students, and professors from around the world. By the end of the weekend, 21 ideas with corresponding pitches were developed in five thematic areas: health needs, education, small businesses, community, and purchasing. We describe how the community was rapidly rallied, and we discuss the key learning outcomes of this spontaneous entrepreneurial endeavor. We provide evidence from participants and mentors that showcases the value of the time-compressed virtual idea blitz in accelerating social entrepreneurial action. We offer practical guidance to academic, community, and professional institutions that would like to replicate or build upon our approach to stimulate the formation of community and to coordinate efforts to thwart the ongoing threat of COVID-19, as well as other societal challenges that might emerge in the future.