DONDE ANDERSON

The first step in protein expression is ligating (inserting) the plasmid into a vector that contains genes specific for protein expression. The vector Donde is using is called pTYB2. This has genes which when transformed into the host will express the protein but also contains genes which will aid in selecting the appropriate cells which have the modified phytochrome
	The diagram here is of the vector pTYB1 which is an analog of pTYB2. This vector contains some specialized genes, seen as dark arc bands, which will help Donde in isolating the mutated phytochrome.
	MCS	Multi-Cloning Site- The position where the plasmid is inserted into the vector. The lines pointing the vector are different endonucleases and the sites where each one cuts the vector
	CBD	Chitin binding domain- This site contains a gene for a protein which will bind with chitin. Chitin (a polysaccharide) is used in a chromatography column to bind with the mutated protein complex.
	LacZ	b-galactosidase-Reporter genes are used in vectors to easily identify those cells where the protein is expressed. LacZ is a gene which synthesizes an enzyme that breaks down a specific sugar molecule. Cells that do not possess the recombinant gene produce a blue color whereas in the nutrient medium, whereas those cells that have expressed the gene produce the enzyme that breaks down the sugar and is seen as clear or white.
The faint blue spots are representative of those cells that do not contain the insert/vector. Some dots on the plate are white or clear, which means they possess the LacZ gene and also the mutated plasmid.

The ligation processes is performed in vitro, but the plasmid (pTYP2 and insert) next has to be transformed into a host cell, E. coli or P. pastoris. This involves seeding the cultures, adding different materials to the nutrient medium which will help in the screening processes, and finally incubating. See Donde's Subcloning information on some of these processes.

Once the cells have been incubated and screened by identifying the cultures that appear clear, the protein must be isolated. This is done by lysing the cell using a sonicator (amplified sound waves), centrifuging the cell components and then filtering using a chitin column.

Jeong-Il is demonstrating the sonicating process to Donde. The sonicator will break up the cell wall and membranes to produce a mixture of cell components. This method doesn't use enzymes which could cleave the mutated phytochrome protein so it is preferred over enzyme digestion.

The columns to the right contain a polysaccharide, chitin. Chitin is the same molecule that is found in exoskeletons of insects, spiders and many crustaceans. This polysaccharide will act as a binding agent for the new mutated protein complex. The complex contains a polypeptide segment called Chitin Binding Protein, which is produced from the Chitin Binding Domain on the vector. This protein will adhere to the chitin and allow all other materials to wash through the column, thus selecting only the mutated protein.

	This diagram shows a representation of the mutated protein complex that is formed from the transformed vector.
		The top caption shows the Vector containing the DNA insert, MCS and CBD.
		The vector, through cloning and expression, will produce the protein complex. This complex contains the targeted protein (black segment), an intein, and the chitin binding protein.
		The third caption shows the column chromatography process where chitin is used as the gel. Note to the right that the chitin binding protein and chitin are bonded together. This allows all other cell components to be washed through the gel.
		The last step is to cleave the target protein from the chitin binding protein and intein. This is done by using a chemical called DTT, Dithiothreitol. This cleaves the peptide bond between the mutated phytochrome and the intein.

The final process associated with isolation of the mutated phytochrome is to verify the protein by SDS-PAGE and Western blot analysis.