Abstract

The availability of complete sequences for the genomes of many organisms changes the researcher's attention from genome sequencing to finding the function of gene products, the proteins. The three dimensional (3D) structure of a protein is helpful to understand its function, because tertiary structure is more conserved in evolution than sequence. The 3D structures can be found quickly by using high-throughput (HT) methods for protein expression, purification, characterization and structure determination. This process is very challenging in every step. Therefore we selected nearly 200 cancer related proteins as targets to see the difficulties in trying to determine the web predicted structural properties that make the protein easy to produce and crystallize using current technologies. Selection of targets, gene carriers or vectors and the expression strains of Escherichia coli K12-MG1655 (EC) are very important to obtain soluble expression clones and pure protein for crystallization and structure determination. Identification of protein function based on 3D structural analysis comes after the structure determination. We described a new calculation method called frame method to characterize the proteins in the mu-crystallin fold family. Two functionally characterized proteins, ornithine cyclodeaminase (OCD) and alanine dehydrogenase (AlaDH), are used as frames in order to annotate the activity of OCD like 1 from thermophilic Sulfolobus solfataricus P2 (Ss) and the human mu-crystallin enzyme. OCD like 1 seems to have OCD activity, but the human enzyme does not, as described in the literature. In the meantime, OCD like 1 expression was tested and purified to test for OCD activity to validate our calculations. We were not able to purify OCD like 1; therefore the determination of catalytic activity of it will require future studies. Crystallization, structure determination and identification of protein function are dependent on each other; therefore we started crystallization studies of Phosphoinositide-specific phospholipase C (PI-PLC) from Streptomyces antibioticus (Sa) to better understand the mechanism of action of the PLC protein family. SaPLC is purified by affinity, gel filtration and ion exchange chromatographies. SaPLC crystals are obtained using the hanging drop crystallization technique at concentrations of 20-24 % polyethylene glycol (PEG) 17500 and 1.5- 2 % 1,4- dioxane for 35-40 mg/mL protein. The main major barrier to success of structure determination is the preparation of well-diffracting crystals for X-ray diffraction studies, which we are currently optimizing.

Notes

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Graduation Date

2005

Semester

Summer

Advisor

Selby, Thomas L.

Degree

Master of Science (M.S.)

College

College of Arts and Sciences

Department

Chemistry

Format

application/pdf

Identifier

CFE0000632

URL

http://purl.fcla.edu/fcla/etd/CFE0000632

Language

English

Release Date

October 2018

Length of Campus-only Access

None

Access Status

Masters Thesis (Open Access)

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