Shin Lab 

Welcome to the Shin Lab homepage at the University of Toronto!  If you are reading this page, you are probably interested in science and research.  Excellent.  If you have more questions, please contact Jumi Shin at jshin@utm.utoronto.ca or (905) 828-5355.

Our research started at the University of Pittsburgh in 1995.  We moved to the U of T in Summer 2002.  Here, at the Mississauga campus (about 15 miles/23 km from downtown Toronto), we have a large, highly interactive community of researchers at the interface of chemistry and biology, including groups in bioinformatics, biotechnology, and biophysics.  The environment here is excellent for learning and doing cutting-edge science.  This campus is committed to interdisciplinary sciences, as can be seen by the rapidly expanding infrastructure (lots of new construction), programs (for instance, the new Biotech programs), and hiring of many new faculty with interdisciplinary research interests (like me).

In our group, we take a problem-solving approach to our work in protein design.  We study how proteins, Nature's universal scaffold for molecular recognition, interact with specific ligands when there are so many to choose from in the cell.  In order to gain an understanding of this large problem, we use a diverse array of tools from molecular biology, biochemistry, organic and physical chemistry.  We apply our knowledge to protein design, in particular, design of proteins that target specific DNA sites.  In this way, we may be able to design new drugs that target specific diseases with fewer side effects. 

Some examples of experiments are shown below.  Click on the photos to see enlarged data graphics.

DNA Synthesis, Cloning, and PCR.  We construct genes that code for our designed proteins.  These genes can be synthesized on a DNA synthesizer, by using polymerase chain reaction (PCR), or a combination of both.  Genes are then cloned into plasmid DNA.  Bacterial cells are transformed with these engineered plasmids, and these cells become mini factories for our proteins.  The graphic outlines the basic steps we use to construct a gene using DNA synthesis, PCR, cloning, and transformation. 

Protein Expression and Purification.  After growing protein in transformed cells, the cells are lysed and the contents purified.  This can be seen in the SDS-PAGE gel showing the purification of protein through different steps:  molecular weight markers (Lane 1), crude cell lysate (Lane 2), after purification on a metal-ion affinity column (Lane 3), and after size-exclusion chromatography (Lane 4). After the final purification shown in Lane 4, note the purity of protein (denoted by the red arrow).

Chromatography:  HPLC, FPLC, ion-exchange, size-exclusion.  Typically, more than one type of chromatography is needed to purify our proteins.  After metal-ion affinity chromatography, we commonly use reverse-phase HPLC or size-exclusion chromatography.

Gel Electrophoresis.  DNA is separated on low-resolution agarose gels or high-resolution polyacrylamide gels (PAGE).  Proteins are visualized on PAGE gels followed by immunoblot assay.  A high-resolution DNase I footprinting gel is shown; if our protein (or ligand) binds to a specific DNA site, it will protect that site from random DNase I cleavage.  This can be seen by the empty space or "footprint" left by our proteins binding specifically to the AP-1 and ATF/CREB sites on a ~650 base-pair fragment of DNA.  Footprinting is an especially stringent technique for evaluating specific binding of ligands to DNA.  From this data, we conclude that our proteins can specifically bind to desired DNA sites.

Circular Dichroism.  We are designing a-helical proteins.  This particular structure can be measured by CD; a-helices display a double minima at 208 nm and 220 nm.  The CD shown here confirms that we have the desired a-helical structure for all four of our proteins.

Fluorescence Anisotropy.  In an anisotropy titration, we add aliquots of protein to fluorescein-labeled DNA in a cuvette.  As protein concentration increases, binding to DNA increases and increases the anisotropy of the fluoresceinated DNA:  i.e. as the protein binds, the tumbling motion of DNA in solution slows down.  This experiment gives us information on the thermodynamics of protein-DNA complexation.  We generate a binding isotherm which provides us with the free energy of binding in our system.  This is a terrific experiment; you can do a titration and fit the data on the computer, and within a couple of hours, you have your free energy.

Mass Spectrometry.  Measuring the masses of your proteins can give you valuable information about whether or not you have the expected protein.  Often, post-translational modifications occur on a protein, so sequencing proteins using MS is very useful.  We have developed techniques for using MALDI-TOF mass spectrometry to gain masses of extremely hydrophobic proteins.  The graphic shows MALDI-TOF of one of our proteins:  the cystine dimer, matrix adduct, mercaptoethanol adduct, and protein proteolyzed at the methionine amino terminus are detected.  We also use electrospray ionization MS for studying protein fragments.