In a recent Jones Seminar at the Thayer School of Engineering at Dartmouth, Karl E. Griswold, Assistant Professor of Engineering, discussed his recent research into applied biocatalysts.
Griswold began his lecture with an explanation of the basics of protein engineering. Proteins are long strings of amino acids that coil together to form complex polymer molecules. These molecules are then able to perform a wide array of metabolic functions from providing structure to catalyzing reactions.
Outside of the body, this diversity of protein function can be applied to solve various problems. Griswold gave various examples of applied protein technology that exist today, including dish cleaners, gasoline, performance enhancing drugs, recycled paper, and cheese production.
Griswold then transitioned to discuss the main goals of his specific research group. He hopes to use protein engineering to address the challenge of drug resistant pathogens. Scientists often develop drugs, or antibodies, to fight certain strands of pathogens. However, pathogens are able to develop resistance to these antibodies over time, often more quickly than new antibodies can be developed. Griswold and his research group hope to use protein engineering to develop antibodies that will be less prone to resistance from pathogens.
More specifically, the group aims to redesign human lysozyme to better fight pathogens. Lysozyme is a defense enzyme naturally produced by the immune system. It works by breaking down the peptidoglycan cell walls found in bacteria. However, the enzyme’s effect can by greatly diminished by factors in the environment and by inhibitors produced by some bacteria. Griswold’s group looks towards protein engineering to increase effectiveness of lysozyme in spite of inhibiting conditions.
Griswold’s research group hopes to improve lysozyme function in an environment of negative anions. In patients with cystic fibrosis, organic polyanions like alginate, DNA, F-actin, mucin, and P. aeruginosa spill into the intercellular spaces of the lungs and intestines. Human lysozyme is a positively charged molecule and therefore does not normally work efficiently in negatively charged conditions. However, by altering the DNA that codes for lysozyme production, a process known as mutagenesis, researchers have been able to change the structure and charge of lysozyme. However, researchers have experienced some difficulty in retaining the normal function of lysozyme with an altered structure or charge. In order to find a mutation of lysozyme that is both functional and resistant to negative anions, researchers created a “combinatorial library” of possible mutations and tested the viability of each mutated strand of lysozyme through trial and error. Using this method, Griswold’s research team was able to identify a strand of mutated lysozyme (labeled 2-3-7) that was three times more resistant to alginate, 20 times more resistant to F-actin, and 40 times more resistant to DNA than the wild type lysozyme. In addition, the engineered strand was found to function twice as efficiently as the wild type under normal conditions.
Using a similar approach, Griswold’s group was also able to address the ability of some bacteria to produce inhibitors that block lysozymes from functioning properly. More specifically, the research looked into the “Ivy” family of inhibitors often found in Gram-negative bacteria. These inhibitors work by binding to lysozymes and physically blocking their active site clefts. By creating another combinatorial library and testing using trial and error, Griswold was able to create variants of lysozyme with a different active site cleft shape, while maintaining the normal function of lysozyme. These variants of lysozyme had significantly increased immunity to the effects of Ivy inhibitors; they were able to maintain between 50 and 70 percent efficiency at 400nm concentrations of Ivy inhibitor, a concentration level that would normally completely inhibit wild type lysozyme.
Professor Griswold concluded by emphasizing the future applications of combinatorial protein engineering, saying: “biocatalysts are the next generation antibiotics.”