2006 USDA-supported Students -Department of Biological Sciences - Carnegie Mellon University

2006 USDA-supported Students

Leslie Ann AlexisLeslie Ann Alexis, Hunter College
Mentor: Dr. Peter Berget

The Role of Cysteine Residues in Single Chain Antibodies
Cancer treatment and the treatment of other diseases such as Alzheimer's may be improved with increase understanding of signal transduction pathways and the stepwise mechanism of other biological processes within a cell. A biosensor is a molecule that makes this possible. A potential biosensor is a single chained (scFv1) antibody with the fluorescent dye thiozole orange (TO1- 2P). TO1- 2P in aqueous solution exhibits low levels of fluorescence but interacts with scFv1 to cause higher levels of fluorescence. scFv1 is expressed on the cell surface of Saccharomyces cerevisiae via the alpha agglutinin receptor . scFv1 contains a variable light and a variable heavy chain joined together by a linker. Each chain of the scFv1 contains two cysteine residues. When in an oxidizing environment these cysteine residues form disulfide bridges, which aid in the proper folding of the protein. However, in a reducing environment these disulfide bonds may not form, resulting in misfolding of the scFv1. Thus intracellular expression of these proteins may result in inactive biosensors. Site directed mutagenesis was used to change each of the cysteine residue pairs into the hydrophobic amino acids alanine and valine respectively. Such amino acid substitutions have been shown to result in a substitution of hydrophobic interactions that mimic the disulfide bonds in other scFvs. The scFv1 in which the C- terminal cysteine was changed to alanine was expressed on the surface of a S. cerevisiae strain. Flow cytometry analysis showed that the protein was expressed on the surface of S.cerevisiae . Future experiments will be done to study the effects of this mutation on the proteins interaction with the TO1- 2P dye, as well as, the effect on changing the other cyteine residues on protein folding.

Yetunde JunaidYetunde Junaid, Hunter College
Mentor: Dr. Bruce Armitage

7-[(2-Aminoethyl)amino]-1-methylquinolinium iodide: A Fluorescent Dye that Absorbs Around 400 nm.
There is little tendency for cells to absorb light in the region close to 400 nm. Therefore, synthesizing fluorescent dyes that absorb at such region would very likely eliminate some background fluorescence and increase the sensitivity of various imaging and detection method. 7-[(2-Aminoethyl)amino]-1-methylquinolinium iodide is a fluorescent dye that not only absorbs at 404 nm, but is also known to have high fluorescence quantum yield, thermal and photochemical stability, and large Stokes shifts ( l em   489 nm). The goal of this research was to synthesize this dye and an analogue that can be used in bioimaging applications. The synthesis of the dye followed a literature procedure. A mixture of 5-fluoroquinoline and 7-fluoroquinoline isomers previously synthesized in the lab was reacted with iodomethane in the presence of methanol to yield the N-alkylated isomer mixture. This was washed with ethyl ether, and the desired 7-fluoro-1-methylquinolinium iodide recrystallized from 96% ethanol. This intermediate in turn was reacted with ethylenediamine to yield 7-[(2-Aminoethyl)amino]-1-methylquinolinium iodide. 1 H- NMR was done at each step to confirm the synthesis of the expected intermediates and at the end to make sure the expected end product was synthesized. The fluorescence as well as UV-vis absorption spectra of the dye in different media were measured. A biotinylated derivative was also prepared in order to study its possible use in selection processes and as a label for flow cytometry and confocal microscopy.