BICH 409: Protein Purification (1)

What is needed to explore protein structure and function?

protein must be purified

How many proteins can be in a singular cell?

thousands of proteins

What % of the protein of interest is the cells' dry weight?

less than 0.1%

How do biochemists isolate proteins?

differences in physical and chemical behavior of proteins

What are some of the differences within proteins?

solubility

ionization

size

polarity

chemistry

Where does the protein of interest come from?

animal tissue

microorganism

What is an example of the vector of gene of the protein of interest?

E.Coli

What % of the protein of interest can be a part of the E coli cell?

40%

What is the first step of protein purification?

lysing the cells

How can animal tissue be lysed?

osmotic lysis: put tissue in a hypotonic solution

What is sonication?

high frequencies of sound breaking cell walls

What is a french press?

high pressure forces cells through pinhole opening causing cells to lyse open

What enzyme breaks down bacteria's cell walls?

lysozyme

What comes after lysing the cells?

centrifuging the cell debris

What is needed to protect the protein of interest in centrifuging?

protease inhibitors

What degrades proteins?

serine and cysteine proteases

What is EDTA used for in centrifuging cells?

complexing all of the metals in solution: metalloproteases

What's unique about the crude lysate?

must be kept cold: place on ice

What is the 3rd step in protein purification?

assay: means of quantitatively detecting its presence

What are the simplest and most accurate assays?

spectroscopic assays