Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

PURIFYING PROTEINS
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In other cases, an entire protein is used as the recognition tag. When cells
are engineered to synthesize the small enzyme glutathione S-transferase (GST)
attached to a protein of interest, the resulting fusion protein can be purified from
the other contents of the cell with an affinity column containing glutathione, a
substrate molecule that binds specifically and tightly to GST.
As a further refinement of purification methods using recognition tags, an
amino acid sequence that forms a cleavage site for a highly specific proteolytic
enzyme can be engineered between the protein of choice and the recognition tag.
Because the amino acid sequences at the cleavage site are very rarely found by
chance in proteins, the tag can later be cleaved off without destroying the purified
protein.
This type of specific cleavage is used in an especially powerful purification
methodology known as tandem affinity purification tagging (TAP-tagging). Here,
one end of a protein is engineered to contain two recognition tags that are separated
by a protease cleavage site. The tag on the very end of the construct is chosen
to bind irreversibly to an affinity column, allowing the column to be washed
extensively to remove all contaminating proteins. Protease cleavage then releases
the protein, which is then further purified using the second tag. Because this twostep
strategy provides an especially high degree of protein purification with relatively
little effort, it is used extensively in cell biology. Thus, for example, a set of
approximately 6000 yeast strains, each with a different gene fused to DNA that
encodes a TAP-tag, has been constructed to allow any yeast protein to be rapidly
purified.
Purified Cell-free Systems Are Required for the Precise Dissection
of Molecular Functions
Purified cell-free systems provide a means of studying biological processes free
from all of the complex side reactions that occur in a living cell. To make this possible,
cell homogenates are fractionated with the aim of purifying each of the individual
macromolecules that are needed to catalyze a biological process of interest.
For example, the experiments to decipher the mechanisms of protein synthesis
began with a cell homogenate that could translate RNA molecules to produce
proteins. Fractionation of this homogenate, step by step, produced in turn the
ribosomes, tRNAs, and various enzymes that together constitute the protein-synthetic
machinery. Once individual pure components were available, each could
be added or withheld separately to define its exact role in the overall process.
A major goal for cell biologists is the reconstitution of every biological process
in a purified cell-free system. Only in this way can we define all of the components
needed for the process and control their concentrations, which is required to work
out their precise mechanism of action. Although much remains to be done, a
great deal of what we know today about the molecular biology of the cell has been
discovered by studies in such cell-free systems. They have been used, for example,
to decipher the molecular details of DNA replication and DNA transcription, RNA
splicing, protein translation, muscle contraction, and particle transport along
microtubules, and many other processes that occur in cells.
Summary
Populations of cells can be analyzed biochemically by disrupting them and fractionating
their contents, allowing functional cell-free systems to be developed. Highly
purified cell-free systems are needed for determining the molecular details of complex
cell processes, and the development of such systems requires extensive purification
of all the proteins and other components involved. The proteins in soluble
cell extracts can be purified by column chromatography; depending on the type of
column matrix, biologically active proteins can be separated on the basis of their
molecular weight, hydrophobicity, charge characteristics, or affinity for other molecules.
In a typical purification, the sample is passed through several different columns
in turn, with the enriched fractions obtained from one column being applied
to the next. Recombinant DNA techniques (described later) allow special recognition
tags to be attached to proteins, thereby greatly simplifying their purification.