Anna Katz and Dr. Brad Bundy, Chemical Engineering

Introduction

The purpose of this project was to examine the phenomenon of eukaryotic myristoylation in a prokaryotic cell-free environment. Basically the idea is that eukaryotic cells such as those within humans have a more robust system for synthesizing protein but it is cheaper, faster, and more easily controlled to synthesize protein in bacteria cells. The problem, then, with using bacteria is that it lacks the ability to perform such functions as post-translational modification; in other words, once the protein has been created through RNA transcription separate enzymes add functional groups to the protein. One such modification is myristoylation, which uses the enzyme N-myristoyltransferase (NMT) to attach a fatty acid to the end of the protein. Bacteria such as E. Coli do not naturally have this enzyme present and so are unable to perform myristoylation. Our research has been an attempt to show that myristoylation is possible in a system composed of prokaryotic cell extract with the addition of NMT plasmid.

Process

The first step was a look at currently published literature to see what others have done in this area. Many have experimented with myristoylation though none had attempted it in a cellfree bacteria-based system. In the course of previous experimentation it was established that a basic code of 6 amino acids were necessary in the structure of the protein to ensure myristoylation. We decided to insert this code into a well-known protein that Dr. Bundy had experimented with, namely Green- Fluorescent Protein (GFP). The advantages of this protein are one: its known success within the given E. Coli based system, and two: its fluorescence makes it easily detected. The second step of the project, then, was to perform the cloning necessary to insert this gene into a GFP plasmid. This process actually took much longer than we had planned for because of the difficulties involved with cloning. Eventually a system of PCR, ligation, and transformation produced the desired plasmid, as checked by an outside sequencing company. From there it was time to proceed with testing.

In testing the desired goal was to add an acid group to the protein as it was being synthesized in our cell-free environment. The difficulty with this task lay in the detection of the myristoylation. Because the acid group only added 200 daltons we had to find a very precise way to determine if our product was myristoylated or not. Many attempts were made using highperformance liquid chromatography with mixed results that, more recently, have looked promising. Currently we are undergoing further tests with mass spectroscopy that should give us the confirmation we need to know that myristoylation is occurring.

Results/Future Directions

I made a poster documenting our tests thus far and presented it at the American Institute of Chemical Engineers Annual Conference in Salt Lake City, in November of this year. Should we get confirmation of further results we can continue testing to attempt to optimize myristoylation in our system. This could also lead to potential vaccine development for viruses such as foot-and-mouth and rhinovirus that require myristoylation in their coat proteins. In February of next year I will be presenting at the Utah Conference on Undergraduate Research and hope to have more definitive data at that time. Eventually this could lead to a published paper, as myristoylation is a topic often discussed in scientific literature.