Table 5-1

Protein Purification

• Essential for characterizing individual proteins (determining their enzymatic activities, 3D structures, etc.)

• Two main aproaches• Classical – from the natural source (e.g. tissue)

• Advantages: natural (modifications, binding partners, etc.)

• Disadvantages: intractable for low abundance proteins (~20,000 types of proteins in the cell!)

• Molecular cloning (insert gene into heterologous host and induce cells to make lots of the protein of interest

• Advantages: abundance, purification tricks, genetic variations easy

• Disadvantages: may lack natural modifications, partners, etc.

Text, Figure 5-2

Overexpressing a protein faster than the host cell (E. coli) can fold it leads to aggregated protein in an inclusion body. After isolating inclusion bodies (by cell disruption and centrifugation), often the desired protein can be unfolded and successfully refolded. [Not necessarily a desirable approach]

Protein Overexpression

Figure 5-4

Tryptophan has a strong absorbance at 280nm. This is useful for detecting the presence of proteins during chromatography, and in estimating protein concentration. To be accurate, one needs to know the amino aid composition of the protein, especially how many Trp’s there are. This is usually know from genetic data.

Keeping track of proteins during purification

Text, Figure 5-4

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Properties of proteins that form the basis for various purification strategies

Heat stability precipitation at high Temp

Purification by ammonium sulfate precipitation• Everything has a finite solubility• The solubility of a protein tends to decrease as the

concentration of salt (especially polyvalent) increases• The solubilities of different proteins in ammonium

sulfate are different proteins• Therefore, as you increase the A.S., different proteins

reach their solubility limits and come out of solution (i.e. precipitate) at different points

Increasing ammonium sulfateAdvantages:• Scales-up well• Simple, cheap

Disadvantages:• Not very specific• The protein must be at fairly high

concentration or its solubility limit may not be reached

Text, Figure 5-5

Figure 5-6

Ion-exchange chromatography separates proteins mainly based on charge properties

Anion exchange:• Use a column whose

matrix is positively charged (e.g. quaternary ammonium groups)

• Add protein mixture to column at reasonably high pH (Why?)

• Elute by running through solution with increasing salt concentration (Why does this work)

[Cation exchange chromatography is the reverse]

Text, Figure 5-6

Figure 5-7

Size exclusion or gel filtration chromatography:Separation based on molecular size (of the native complex). Large molecules migrate fastest(!)

Advantages:• Informative (e.g.regarding

native size)• simple

Disadvantages:• Capacity limited• Not very good resolving

power (~ factor of 2 in size)

Text, Figure 5-7

Affinity chromatography: separates proteins based on specific binding property

Examples:Attach ligand that the protein binds to the

column matrix

Attach an antibody to the column that recognizes the protein

Use molecular cloning techniques to add a short sequence tag to the protein of interest, and make a chromatography matrix that binds the tag

• Super powerful, specific• General, doesn’t require any special

property of the protein• Most common: metal (e.g. Ni) attached

to column, His6 tail attached to protein• Terminal tail can be cut off at the end

Text, Figure 5-8

Figure 5-8

Monitoring the progress of purification, and knowing if you’ve got the right protein:

Gel electrophoresis

Figure 5-9

SDS-PAGE (polyacrylamine gel electrophoresis):

• Often used for monitoring progress of purification

• Protein chains are denatured by the SDS detergent

• The SDS is (-) charged and tends to bind similarly (i.e. proportionally) to all proteins

• Then, negatively charged electrode in electrophoresis drives proteins downward in the gel (driving force is proportional to protein size, but frictional force is more strongly dependent on size, so large proteins migrate more slowly)

• A lane containing a ‘ladder’ of molecular weight standards run at the same time, makes it possible to estimate the MW of the protein chains

• Often used for monitoring progress of purification

• Protein chains are denatured by the SDS detergent

• The SDS is (-) charged and tends to bind similarly (i.e. proportionally) to all proteins

• Then, negatively charged electrode in electrophoresis drives proteins downward in the gel (driving force is proportional to protein size, but frictional force is more strongly dependent on size, so large proteins migrate more slowly

• A lane containing a ‘ladder’ of molecular weight standards run at the same time, makes it possible to estimate the MW of the protein chains

SDS-PAGE (polyacrylamine gel electrophoresis):

Text, Figure 5-10

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Coomassie: an example of a protein specific dye that is used widely to detect protein bands in gels

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An example of SDS-PAGE from the lab, showing the progress of protein purification

Crude mixture (e.g. whole cells)

Single fraction after Ni-column chromatography (and other purification

Molecular weight standards

kDa

Figure 5-11

1. Run one kind of electrophoresis2. Cut out lane 3. Rotate, and run second type of electrophoresis

An example of 2D gel electrophoresis: tremendous resolving power

Text, Figure 5-11

Using ELISA to detect the presence of a particular protein of interest

You would typically run an ELISA experiment (or other activity based assay) on each of the tubes or ‘fractions’ coming off a chromatography column

Text, Figure 5-3