Biology Reference
In-Depth Information
stabilizing hydrophobic effect driven by water structure (Chapter 3). Unfortunately, chaot-
ropic agents can also denature proteins, and the enormous concentrations employed makes
their complete removal difficult, limiting their usefulness.
The methods discussed in Chapter 12 for isolation and purification of membrane fractions
are also applicable for the isolation and purification of membrane integral proteins. Many of
the protein isolation methods are based on centrifugation and affinity chromatography. The
major difference is that extraction of an integral protein from its membrane in non-denatured
form generally involves the use of mild detergents. Obtaining a non-contaminated mem-
brane fraction in high yield greatly enhances the chance of subsequently obtaining a partic-
ular protein in pure form. For example, if the objective is to extract and purify cytochrome
c oxidase, a first step will involve obtaining an intact mitochondria by simple differential
centrifugation.
Moderate concentrations of mild detergents are used to compromise membrane integrity
and facilitate the extraction of integral proteins in water-soluble, non-denatured form. The
extracted proteins are found in mixed micelles composed of the protein, membrane phospho-
lipids, and the detergent. The effect of detergent levels on membranes is concentration-
dependent.
Detergent/Lipid ratio
Effect on membrane
0.1:1 to 1:1
Bilayer remains intact. Some proteins extracted.
1:1 to 2:1
Bilayer solubilized into mixed micelles.
10:1
All lipid
protein interactions are replaced by
e
detergent
protein interactions.
e
The membrane solubilization buffer should contain enough detergent to provide greater
than one micelle per membrane protein in order to assure that individual proteins are iso-
lated in separate micelles. The required amount of detergent will depend on the detergent's
physical properties (CMC, HLB, aggregation number) as well as the aqueous buffer pH, salt
concentration, and temperature.
The complex solution containing many membrane proteins (mostly located in mixed
micelles), can then be passed over a lectin or antibody affinity column (discussed in
Chapter 12). Only the selected protein binds to the column and the remaining, un-bound
proteins are discarded. The bound protein can then be removed by addition of excess
free sugar for a lectin affinity column or by altering the pH or salt concentration for an anti-
body affinity column.
If the desired protein is hard to isolate or is present in low amounts, recombinant DNA
technology may be employed. The protein may be expressed in large quantities in another
cell type. Also the newly expressed protein can be tagged by something, often a poly-histidine
tag (His-tag) that can greatly facilitate the protein's isolation.
The His-tag technique usually involves engineering a sequence of 6
8 histidines to either
the C- or N-terminal of the protein [8,9] . The poly-histidine tag binds strongly to divalent
metal ions like nickel or cobalt. Therefore, if a complex mixture of proteins is passed through
e
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