The Construction Of Bacterial Expression Plasmids With Human Cdnas Encoding Neurofilament Proteins
THE CONSTRUCTION OF BACTERIAL EXPRESSION PLASMIDS WITH HUMAN CDNAS ENCODING NEUROFILAMENT PROTEINS
JOSEPH D. KLAVITTER
Advisor: Dr. Sharon Ackerman
Major: Biochemistry and Molecular Biology
Degree: Master of Science
Neurofilaments (NFs) are the most abundant cytoskeletal components of large myelinated axons from adult central nervous system (CNS) and peripheral nervous system (PNS) where their major function is to control axonal caliber (25). The genetic composition of intermediate filaments consists of at least 67 genes that encode functional intermediate-filament proteins, which makes this gene family one of the largest in the human genome (26). Mammalian NFs are composed of three polypeptides, classified as light (NF-L, 61 kDa in humans), medium (NF-M, 90 kDa) and heavy (NF-H, 110 kDa) (27). The diameter of IFs (10 nm) is “intermediate” between microfilaments (6 nm) and myosin filaments (15 nm) (6). Very little is known at the molecular level about how the normal distance between NFs is set and maintained; although the idea that phosphatase may be linked to neurofilament compaction (NFC) is a prevalent viewpoint in the field.
This research project was designed to provide new information about the structure/function relationships in neurofilaments (NFs). Important questions relevant to neurofilament biology were identified that could be addressed in biochemical experiments with purified recombinant NF proteins that are produced in Escherichia coli; through the development of a bacterial expression system for the production of NF-Lrod:NF dimers as recombinant proteins for use in further in vitro experiments. The ultimate objective is to produce soluble NF-Lrod:NF-L and NFLrod:NF-M dimers that would one-day lead to the crystallization of neurofilament proteins and ultimately reveal new methods to treat NFC and neurodegenerative diseases.