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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In olden days, 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.  Purification based on solubility and charge.

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

Step 1:  Obtain 25 lbs of fresh chicken hearts on ice from local slaughter-house.

Step 2:  Cut tissue in small sections, grind tissue in a commercial meat grinder, extract with 14L cold distilled water.

Step 3:  Filter cell and connective tissue debris from extract by passing thru double layer of cheese cloth.

Step 4:  Centrifuge using 1L containers and large bucket rotor International centrifuge at 1300 x g for 30 minutes.  Pool supernatant. Assay.

Step 5:  Adjust pH to 7.0, add solid ammonium sulfate to give 70% saturation of AS, maintain pH ~ 7 by adding dilute ammonium hydroxide as needed.  Let stand for 2-4 hours, filter overnight on fluted filter paper.  Scrape ppt. off filter paper and suspend in 1 L of cold distilled water.  Centrifuge.  Assay.

Step 6:  Dialyze soluble fraction against two 20 L changes of 0.005M Tris buffer, pH 7.0 for a total of 12 hours.  To the solution, add solid AS to 25% saturation.  Centrifuge, remove ppt.  Add more AS to 60% saturation, leave standing for 1 hour, spin at 18,000 x g for in a Sorvall refrig. Centrifuge, save pellet.  Dissolve pellet in 300 mL of 0.005 M Tris buffer, pH 7.6.  Dialyze against two 20 L changes of 5 mM Tris, pH 7.0 for 8 hours each, centrifuge.

Step 7:  Add precooled (-15 deg) acetone slowly to dialysate to ppt protein at 25%/(v/v).  After 10 minutes, cent. – discard ppt.  Add more acetone to 50% (v/v), leave for 20 minutes at 0^oC.  Collect ppt. At 18,000 x g for 30 min., then extract with 200 mL cold Tris buffer, pH 7.6, cent., discard ppt.

Step 7:  Add supernatant to DEAE cellulose column.  Elute with a gradient of 2 L each of 0.005 M Tris, pH 7.0 and same buffer with 0.20 M NaCl.  Combine fractions with LDH activity.  Assay.  Expected yield 50-60 %.

Step 8:  Re-crystallization - Ppt. with saturated AS, redissolve in a minimum of buffer, centrifuge, add AS slowly with stirring to 30% saturation.  Wait about 1 day, centrifuge to harvest enzyme.  Assay.  Yield ~ 30%, 100X purification.

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 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. Size - Dialysis  (figure)

3. Column chromatography - Separation by charge / size / or affinity

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

    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--negatively charged: phosphocellulose, heparin sepharose, S-sepharose
• cationic--positively charged: 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

   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

    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)

• method of separation of charged molecules
• movement of charge particles in an electrical field
• electrophoretic mobility reflects both charge and size/shape
• Many kinds of electrophoresis: usually have a solid medium e.g., paper, gel, TLC plate
• gel electrophoresis used to separate large molecules: DNA, RNA, protein
• a "gel" is like very stiff JELLO
• acrylamide forms polymers that can be cross-linked to varying degrees in a chemical process
• the cross-linking determines the size of "holes" that the molecules pass through

• apply electrical current across the medium, Anode (+) and Cathode (-)
• positively charged molecules move to the Cathode
• negatively charged molecules move to the Anode
• proteins depending on their charge will move in either direction,

• SDS is a detergent--amphipathic, has both hydrophobic and hydrophilic characteristics
• hydrophobic tail of SDS interacts with hydrophobic side chains of amino acids
• number of SDS molecules bound is proportional to the number of amino acids
• 1 molecule of SDS bound per ~2 amino acids
• SDS overwhelms any inherent charges and effectively turns the protein into a polyelectrolyte
• SDS also disrupts tertiary structure
• b-mercaptoethanol is also included to reduce disulfide bonds and destroy intra-chain cross-linking
• protein mobility in SDS PAGE is proportional to protein size