The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory exercise uses size exclusion chromatography (SEC) and ion exchange (IEX) chromatography to separate a mixture of four different proteins. Students use an SEC chromatogram and corresponding SDS-PAGE gel to understand how protein conformations change under different conditions (i.e. native and nonnative). Students explore strategies to separate co-eluting proteins by IEX chromatography. Using either cation or anion exchange, one protein is bound to the column while the other is collected in the flow-through. In this exercise, undergraduate students gain hands-on experience with experimental design, buffer and sample preparation, and implementation of instrumentation that is commonly used by experienced researchers while learning and applying the fundamental concepts of protein structure, protein purification, and SDS-PAGE. V C 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):60-68, 2017.
Changes to the 2015 ACS Guidelines and Evaluation Procedures for Bachelor's Degree Programs included mandatory instruction in polymers and macromolecules for the ACS‐certified degrees. To satisfy this mandate, we have begun developing a series of undergraduate laboratory exercises involving the synthesis, degradation, and characterization of poly(aspartic acid) (PAA). PAA, a biodegradable, water‐soluble synthetic polymer, could potentially replace commonly used poly(acrylic acid), which is a non‐biodegradable polymer that possess environmental risks. In this module of experiments, PAA is prepared in the organic chemistry laboratory course by polymerizing aspartic acid to form poly(succinimide) as an intermediate, which is converted to PAA by base induced ring‐opening. The synthetic PAA, composed of both β‐amide (70%) and α‐amide units (30%), is then transferred to the biochemistry laboratory course for enzymatic biodegradation using two enzymes, PAA hydrolase‐1 (PAAH‐1) and PAA hydrolase‐2 (PAAH‐2). A proposed mechanism suggests that PAAH‐1 hydrolyzes β‐β amide linkages, resulting in oligo (aspartic acid), wherein PAAH‐2 completes the degradation process by cleaving the oligo (aspartic acid) units into monomeric aspartic acid. Current progress on the development of this biochemistry laboratory exercise including overexpression of recombinant PAAH‐1 and PAAH‐2, purification, and characterization will be presented.Support or Funding InformationDUE 1611988
Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved β-sheet flanked by two clusters of α-helices. Within the CDCs, these helices refold into two transmembrane β-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.