BCEM 393 Chapter Notes - Chapter 5: Affinity Chromatography, Isoelectric Focusing, Differential Centrifugation
29 May 2018
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Techniques in Protein Biochemistry
5.1 The proteasome is the functional representation of the genome
- The completely sequenced human genome contains 3 billion bases and about 23,000
protein-encoding genes
- This genomic knowledge, however, does not explain which parts are present in different
components or how the parts work together
- The term proteome has been coined to signify a more complex level of information content —
the level of functional information, which encompasses the types, functions, and interactions
of proteins that yield a functional unit
- The term proteome is derived from proteins expressed by the genome
- The genome provides a list of gene products that could be present, but only a subset of these
gene products will actually be expressed in a given biological context
- The proteome tells us what proteins are functionally present
- The proteome is not a fixed characteristic of the cell. Rather, because it represents the
functional expression of information, it varies with cell type, developmental stage, and
environmental conditions, such as the presence of hormones
- Proteins can be enzymatically modified in a variety of ways. And these proteins do not exist
in isolation; they often interact with one another to form complexes with specific functional
properties
- An understanding of the proteome is acquired by isolating, characterizing, and cataloging
proteins. In some, but not all, cases, this process begins by separating a particular protein
from all other biomolecules in the cell
5.2 The purification of proteins is the first step in understanding their function
- It is important to purify a protein in order to understand the protein’s amino acid sequence, its
3D structure and its function in normal and pathological states
- Purification essentially means isolating the protein of interest from the thousands of other
proteins found in the cell
- Proteins Can Be Purified on the Basis of Differences in Their Chemical Properties
- An assay is a test used after each stage of purification to see if the purification is working
- The assay is based on some unique identifying property of the protein
- For enzymes, which are protein catalysts, the assay is usually based on the reaction
catalyzed by the enzyme in the cell
- After determining how much enzyme activity is present and how much protein is present,
we can assess the progress of the purification by measuring the specific activity, the ratio
of enzyme activity to the amount of protein in the enzyme assay at each step of the
purification
- The specific activity will rise as the protein of interest comprises a greater portion of the
protein mixture used for the assay
- Basically, the purpose of purification is to remove all proteins except the protein in which
we are interested. Quantitatively, it means that we want to maximize specific activity
- Proteins Must Be Removed from the Cell to Be Purified
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