Final Report

Over this summer, professor Eric Chang, Hayley Besser, and I have worked on our goal of developing a protein expression system in order to produce cathepsin-L that mimics the catalytic properties of silicatein-alpha. In order to accomplish this, we first tested different DNA constructs and cell types, such as E. coli, in order to determine which expressed our protein the best. To more properly understand the fundamentals of this process, since neither myself or Hayley had made cathepsin-L before, we first expressed a protein that was previously made by professor Chang, lactate dehydrogenase from the ice fish mackerel, or cgLDH. We first purchased a pUC57 vector from a company known as GeneWiz, which contained the cgLDH gene, and tried to express the protein in DH5-ɑ E. coli cells, which were purchased from New England Biolabs. While the vector was effectively compatible with the DH5ɑ E. coli cells, this was ultimately not a beneficial system for producing and isolating the protein of interest. Because of this issue, we were initially not able to isolate cgLDH protein during affinity chromatography, which raised many questions. During our second attempt, we used a pET11a vector with the cgLDH gene, which was purchased from GeneScript, and T7 express E. coli cells, which were purchased from New England Biolabs. This new setup produced the cgLDH protein on the first attempt, however we noticed that we only produced a very small amount when analyzing the samples using a spectrophotometer. In the future, we can optimize the expression conditions in order to increase the amount of cgLDH produced. Now that we have an established system that works, we can apply this to research with cathepsin-L and see if this protein can be expressed using the T7 E. coli cells and the pET11a vector. If so, we can additionally optimize the growth conditions to produce this protein.

Going forward, we will be able to perform site directed mutagenesis on DNA constructs for cgLDH and cathepsin-L. After this, we can then express the site mutated DNA in order to make new forms of the protein of interest that contain enhanced catalytic and structural properties. The main goal is the systematically change cathepsin-L, through the site directed mutagenesis, in order to mutate it to the point where it matches the chemical and physical properties of silicatein-ɑ.

This program provided me a wonderful opportunity to not only sharpen my pre-existing laboratory and research skills and knowledge, but also to gain new ones. Thanks to professor Chang, I have made antibiotic containing plates, performed chemical transformations with E. coli, performed plasmid mini-preps towards DNA analysis, as well as protein purification and isolation through affinity chromatography. I also had the pleasure of working on a scientific research paper that was presented at the 2018 Gordon Research Conference (GRC) on Biomineralization, which was held at Colby-Sawyer College in New London, New Hampshire from July 28th through August 3rd. One of the biggest advantages of this program has been how I was able to perform different molecular biochemistry protocols and reactions hands on, when most students only learn about them in class. Professor Chang guided both Hayley and myself throughout this entire summer, and provided us with invaluable knowledge and techniques that I will carry with me through the rest of my career as a student.

Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Cathepsin-L Post 2

 

This project has ignited my interest in scientific research and I will continue this project, and another one that will focus on the isolation, construction and modification of a specific yeast killer toxin, K28. I have gained key laboratory skills and protocols that I can utilize in my future experiments and classes. The opportunity to truly experience first-hand the molecular biochemistry reactions was enormously valuable. Many students only hear and learn about some of the processes that I was able to perform in the lab. An example of such was affinity chromatography, a procedure that can be followed in order to separate different biochemical molecules from a mixture based on an extremely specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid. This was something that I learned about in my recent cellular and molecular biology class. In addition to this, I also became more experienced in scientific poster creation and presentation. I assisted my group and instructor with putting together a scientific poster that was presented at the 2018 Gordon Research Conference on Biomineralization. This is an exceptionally useful tool to possess because in my career as a science major, I will need to be able to effectively create and present scientific posters, whether they are for conferences or classes.

As is the case in science, obstacles and difficulties were to be expected. We did have trouble initially because the first construct utilized was not useful to express our protein of interest. This construct was the  pUC57 vector that contained the cgLDH gene, which was purchased from GeneWiz, and DH5ɑ E. coli cells, purchased from New England Biolabs. While the vector was effectively compatible with the DH5ɑ E. coli cells, this was ultimately not a good system for producing and isolating the protein of interest. Because of this issue, we were initially not able to isolate cgLDH protein during affinity chromatography, which raised many questions. During our second attempt, we used a pET11a vector with the cgLDH gene, which was purchased from GeneScript, and T7 express E. coli cells, which were purchased from New England Biolabs. This new setup produced the cgLDH protein on the first attempt, however when we checked the approximate concentration using a spectrophotometer, we noticed that we only produced a very small amount. In the future, we can optimize the expression conditions in order to increase the amount of cgLDH produced. Now that we have an established system that works, we can apply this to research with cathepsin-L and see if this protein can be expressed using the T7 E. coli cells and the pET11a vector. If so, we can additionally optimize the growth conditions to produce this protein.

 

Iterative Site-Directed Mutagenesis Towards Directed Evolution of Cathepsin-L

In our research, the process of iterative site-directed mutagenesis will be used in order to direct evolution of Cathepsin-L. This protein is an important lysosomal endopeptidase enzyme, which is important for the initiation of the process of protein degradation. The goal of our research is to select specific sites of the protein to mutate in order to observe the phenotypic outcomes. Once different mutants of Cathepsin-L are created, we will be able to study the difference in the functionality of the protein, such as whether the mutation hindered or enabled the protein’s ability to perform.

I expect to not only sharpen my pre-existing laboratory and research skills, but also gain new ones. It has been such a valuable experience to be able to perform actual techniques that I have only learned about in school. By being able to do things hands on, I have gained a better understanding of many concepts. Throughout this program, I have made antibiotic containing plates, performed chemical transformations with E. coli, performed plasmid mini-preps towards DNA analysis, as well as protein purification and isolation through affinity chromatography. All of these are techniques and processes that are important for  me to understand and be able to do, not only as a student, but as a scientist.