Using BLAST to Analyze ProteinsPart 2: Comparing Fluorescent Proteins

2013 Workshop C: Cloning DNA to Make ProteinsDina N Kovarik, MS, PhD

NWABR

Fluorescent Proteins are Valuable Tools

• Locate proteins in the cell• Track the migration of cells• Reporter of expression

Sister centromeres (green) mark the attachment of microtubules (red) to sister chromatids (blue). Left: Normal. Right: Drug-treated.

Source: http://mct.aacrjournals.org/content/2/5.cover-expansion

Mice expressing GFP under UV light (left & right), compared to normal mouse (center). Source: Wikipedia.

Bioluminescence of the crystal jellyfish, Aequorea victoria

Source: http://www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html

238 amino acid proteins. GFP ribbon diagram. From PDB 1EMA.

Source: Wikipedia

Rainbow of Fluorescent Proteins

Source: Wikipedia. http://en.wikipedia.org/wiki/File:FPbeachTsien.jpg

• “Drawn” with bacteria expressing 8 different fluorescent proteins

• Diversity of Mutations Diversity of Colors

• “mFruits”• mBlueberry (Blue Fluorescent

Protein, or BFP)• mLemon (Yellow Fluorescent

Protein, or YFP)• mGrape1 (Cyan Fluorescent

Protein, or CFP)• Many others, all with similarly

‘fruity’ names…

Diversity and Key Mutations

Research Questions

(1) Is red fluorescent protein (RFP) related to its famous cousin, GFP, or is from a different source entirely?

(2) What other fluorescent proteins, if any, are closely related to RFP?

The cloning and protein purification experiments you have been conducting in the laboratory involve mTomato (related to mCherry), also called red fluorescent protein (RFP).