Protein Expression, Purification and Analysis

To study proteins and their functions, one must first Produce, Extract, and Purify the protein.

Produce - tissue rich in protein / over-expression using cultured cells (see below)

Extract - cell disruption followed by centrifugation

Purification - take advantages of differences in solubility, charge, size, and specificity.

(An assay is needed to monitor the progress of the purification process.)

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Purification based on solubility and charge: Protein purification used to start with a source (organ tissue, plant source, bacteria line) known to be rich in the protein of interest. Often this would involve obtaining many pounds of organ tissue directly from a slaughter house or growing large (30 or 60L) batches of bacterial culture in order to isolate sufficient material for further studies.

Typical protocol for isolation of a mammalian protein (after procedure for chicken heart LDH – H4, Nathan Kaplan et al., JBC, 239: 1753-1761 (1964):

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Modern Methods: Today, the majority of proteins being studied in the laboratory take advantage of more modern tools of biotechnology to produce large quantities of proteins needed for study.

Restriction enzyme--molecular scissors endonucleases--does not require an end (exonucleases) >100 restriction enzymes known names come from organism: EcoRI--E. coli recognize a specific palindromic DNA sequence and cut the DNA palindrome is the same forwards/backwards some leave 3' overhang; 5' overhang or blunt ends overhangs leave--"sticky ends"--even though DNA is cut, can have base-pairing move DNA from one organism to another - "recombinant DNA" put DNA together with DNA ligase use synthetic DNA of desired sequence to "paste" on restriction site if nature did not provide one	methylation protects DNA from restriction enzymes mechanism for bacteria to protect itself from invading phage or other bacterial DNA
Plamids are cloning vectors plasmids are closed circular DNA, with origin of replication--replicated within bacteria to many copies carries a resistance gene--ampicillin, tetracyclin, kanamycin take DNA from one organism, cut with RE, isolate fragment desired from a gel cut a plasmid or phage DNA with same RE put these two DNA fragments together via sticky ends, ligate them closed we have recombinant DNA this is transferred into bacterial cells by electroporation or chemical competence plate on media with antibiotic to kill bacteria that did not take up a plasmid--no proof that your foreign DNA is there, only that the plasmid is there individual colonies contain a single plasmid
How do you know your foreign DNA was inserted? one method: interrupt a gene that is a reporter - b-galactosidase (lacZ) use a substrate for b-galactosidase that when cleaved give a colored compound do this on antibiotic media to select for plasmid induce the gene with a lactose-analog if the gene is intact get blue color--no foreign insert, just plasmid if the gene has an insert (foreign DNA) then the reading frame is thrown off and no b-galactosidase is produced--no color

Purification - take advantages of differences in solubility, charge, size and specificity.

1. Solubility

Example - Ammonium Sulfate fractions (Figure)
Purification Schemes - need for an Assay / chart results

2. Charge: column chromatography - Separation by charge (size or affinity)

a matrix is in a cylindrical holder
buffer flows through the matrix
fractions are collected
separation of biomolecules

Charge: Ion-exchange chromatography

proteins have charges due to amino acid side groups
bind to charged column matrix depending on their charge at a particular pH
anionic matrix--negatively charged (cation exchanger): CMC (carboxymethyl cellulose), phosphocellulose, heparin sepharose, S-sepharose
cationic matrix --positively charged (anion exchanger): DEAE-sepharose, Q-sepharose
elute bound proteins from column based on charge and displacement by salt or pH

High Performance Liquid Chromatography (HPLC)

gravity flow very slow--depends on size and amount of liquid at the top
HPLC used high pressure to force liquid through
special matrixes and columns
fast and sometimes better resolution

3. Size -

i) Dialysis (figure)

ii) Gel Filtration - separation by size

separates on the basis of size, not charge
porous beads--think of golf balls
small molecules go into the holes and get trapped temporarily (Figure)
large molecules are too large to enter the holes and pass on by
exclusion size--depends on the size of the holes
how long the molecules get trapped determines elution order
large out first > medium > small out last (Figure)
choose the size of matrix for the separation needed
Terms: Vtot, Vo, Vpoly, Ve, Kav = (Ve - Vo)/(Vt - Vo)
Plot Kav vs. log MW for known standards and unknown to estimate MW

4. Specificity:

Affinity Chromatography - use of "tagged" proteins to create affinity site - sep. by specificity

column matrix has a ligand that specifically binds a protein

e.g., ATP-agarose

specialty affinity columns for binding recombinant proteins with certain "tags"

6xHis added at N or C terminus--binds Ni++ column
His tag (Figure 1) (Figure 2) (Figure 3) (Figure 4)
other types of "tags"--chitin, glutathione S-transferase (GST).....

from Qiagen website