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Analyze The Data: Protein Purification

Fractions 21-26 contained larger proteins than fractions 27-36 based on their earlier elution from the size exclusion column. The proteins in fractions 27-36 migrated at a lower molecular weight than those in fractions 21-26 on the SDS gel. The observation that various polypeptides co-eluted on both a size exclusion column and ion exchange column suggests they interact and are subunits of a single protein. To determine which fraction contains the molecular motor, ATP would be added to each fraction and inorganic phosphate production measured using a chromogenic reagent, with the fraction producing the most color likely being the most purified motor. Mass spectrometry of trypsin-digested peptides from the 116 kD

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246 views3 pages

Analyze The Data: Protein Purification

Fractions 21-26 contained larger proteins than fractions 27-36 based on their earlier elution from the size exclusion column. The proteins in fractions 27-36 migrated at a lower molecular weight than those in fractions 21-26 on the SDS gel. The observation that various polypeptides co-eluted on both a size exclusion column and ion exchange column suggests they interact and are subunits of a single protein. To determine which fraction contains the molecular motor, ATP would be added to each fraction and inorganic phosphate production measured using a chromogenic reagent, with the fraction producing the most color likely being the most purified motor. Mass spectrometry of trypsin-digested peptides from the 116 kD

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Quan Thieu
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© © All Rights Reserved
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Analyze the Data: Protein Purification

The purification of a microtubule motor protein has been undertaken (see Vale et al.,
1985, Cell 42:3950). A partially purified cell fraction containing the motor protein was
subjected to an additional purification step using gel filtration chromatography. Aliquots
of each fraction eluted from the gel filtration column were subjected to SDS gel
electrophoresis. Fractions 21 to 41 are shown on the gel below, which was stained with
Coomassie blue.

a. What differing properties of proteins likely account for the bands on the gel
observed in lanes 2126 versus those observed in lanes 2736?

b. Fractions 2832 from the gel filtration column were pooled and eluted over an
ion-exchange chromatography. Again, fractions were collected and aliquots of each
were run on an SDS polyacrylamide gel that was stained with Coomassie blue, as
shown below. What can be deduced about the relationship between the polypeptide
that migrates at about 116 kD on the gel and those that migrate at approximately 66
70kD?
c. Molecular motors are ATPases that use energy of hydrolysis of ATP to
generate a motive (movement) force. Hydrolysis of ATP results in the production of
ADP and inorganic phosphate, the latter of which can be detected by chromogenic
methods. How would you determine which fraction(s) obtained from both of the
columns shown above contain(s) the molecular motor?

d. The band that migrates at about 116 kD in fraction 40 is excised from the gel
and is then subjected to partial proteolysis with trypsin. Mass spectrometry analysis
of the peptides derived from this proteolysis suggests that this polypeptide is a
member of the kinesin motor superfamily. Explain how mass spectrometry data
could lead to this conclusion.
Analyze the DataSolution

a. The proteins first to elute from a gel filtration column are the larger proteins
which do not move into the beads that make up the matrix of the column and thus
only experience the void volume of the column. Fractions 2126 contain proteins
that eluted earlier than the proteins in fraction 27-36. Thus the protein in fractions
2126 should be bigger than the protein(s) in fractions 2736. The observation that
the polypeptide observed in lanes 2126 runs at a higher molecular weight on the gel
than do the polypeptides in lanes 2736 also supports the hypothesis that the protein
in fractions 2126 is a larger protein.

b. The observation that these various polypeptides co-elute on both a size exclusion
column and on an ion exchange column may indicate that these polypeptides interact
and are subunits of a single protein.

c. Add ATP to each fraction from each column. Measure the amount of inorganic
phosphate generated in each fraction (a measure of ATP hydrolysis) by adding the
chromogenic reagent that binds inorganic phosphate. The fraction(s) that contain the
molecular motor should show a color reaction product. If the amount of protein in
each fraction is also measured, one can determine which fraction contains the most
highly purified motor.

d. Mass spectrometry of the peptides derived from the digested polypeptide will
reveal its mass-to-charge ratio, which in turn can provide mass information. Analysis
of genomic data bases can be used to determine which genes in the data base encode
proteins that, when digested with trypsin, would give rise to the size distribution of
peptides that were generated experimentally. Such an analysis may be sufficient to
assign the digested polypeptide to a protein superfamily.

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