Nanopores, megatonnes, and milliseconds : exploring engineered peptides as antimicrobial, carbon-capture,and biocatalytic agents

• Main Author: Barbero, Roberto Juan
• Other Authors: Angela Belcher.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2012
• Subjects: Biological Engineering.
• Online Access: http://hdl.handle.net/1721.1/73773

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. === Cataloged from PDF version of thesis. === Includes bibliographical references (p. 122-131). === This work investigates the roles that peptides play in the fields of antimicrobials, surface functionalization, carbon capture, and biocatalysis. The results demonstrate that peptides, sometimes dismissed for their lack of complexity, can have a breadth of applications. First, the killing kinetics of a pore-forming, engineered antimicrobial peptide (CM15) were imaged using a high-speed atomic force microscope (HS-AFM). The fast time resolution of the HS-AFM (13 seconds per image) enabled characterization of the initial stages of the killing of live Escherichia coli cells. The results suggested that the killing process of CM15 is a combination of a time-variable incubation phase and a more rapid execution phase, offering an interesting parallel between antimicrobial-peptide-induced death and mammalian cell apoptotic death. As a follow-up, an engineered peptide (2K1) with high affinity toward oxide surfaces was used to functionalize a diverse set of materials, including titanium dioxide, zinc, and stainless steel. After demonstrating that 2K1 works as affinity tag for small molecules and fusion proteins, a 2K1-CM15 peptide was made in an attempt to develop a single-step, facile antimicrobial functionalization of oxide surfaces. Second, motivated by the role of peptides in mineralization processes, the yeast Saccharomyces cerevisae was engineered to display peptides and proteins that enhanced the capture of CO2 . An industrial-scale CO2 mineralization process was designed using this engineered yeast with an associated cost of $52 per tonne of CO2 . The effect of the engineered yeast on the process was significant - the cost of CO2 capture was decreased by 8.5-13.5%, as compared to a process with no biological components. Finally, M13 bacteriophage (M13 phage) was established as a temperature stable, highlymultivalent biocatalytic scaffold through display of engineered histidine-biased peptides. A protocol for generating histidine-biased peptide libraries displayed on the major coat protein (pVII) of M13 phage was developed. By analogy to known histidine-based active sites, seven sequences were chosen from amongst hundreds of sequenced histidine-biased pVIII peptides. Two demonstrated esterase activity with a ... 170 that matches, and a ... 4 mM that is only 20-fold lower than, that reported for a commputationally designed esterase. === by Roberto Juan Barbero. === Ph.D.