Protein Engineering

The ability to change the amino acid sequence of a protein by altering the sequence of its cDNA is known as protein engineering. This is achieved through the use of a technique known as site-directed mutagenesis. A new cDNA is created that is identical to the natural one except for changes designed into it by the scientist. This DNA can then be used to generate protein in bacteria, yeast, or other eukaryotic cell lines.

The first use of protein engineering is to study the protein itself. A comparison of the catalytic properties of the normal and mutated form of an enzyme helps to identify amino acid residues important for substrate and cofactor binding sites (Chapter 11). This technique was also used to identify the particular charged amino acid residues responsible for the selectivity of ion channels (page 437). Now scientists are using protein engineering to generate new proteins as tools, not only for scientific research but for wider medical and industrial purposes.

Subtilisin is a protease and is one of the enzymes used in biological washing powder. The natural source of this enzyme is Bacillus subtilis, an organism that grows on pig feces. To produce, from this source, the 6000 tons of subtilisin used per year by the soap powder industry is a difficult and presumably unpleasant task. The cDNA for subtilisin has been isolated and is now used by industry to synthesize the protein on a large-scale in E. coli. The wild-type (natural) form of subtilisin is, however, prone to oxidation because of a methionine present at position 222 in the protein. Its susceptibility to oxidation makes it an unsuitable enzyme for a washing powder that must have a long shelf life and be robust enough to withstand the rigours of a washing machine with all its temperature cycles. Scientists therefore changed the codon for methionine (AUG) to the codon for alanine (GCG). When the modified cDNA was expressed in E. coli, the resulting enzyme was found to be active and not susceptible to oxidation. This was excellent news for the makers of soap powder. However, it is always necessary to check the kinetic parameters of a "new" protein produced from a modified cDNA. For subtilisin (met222) the KM (page 244) is 1.4 x 10-4 M while for subtilisin (ala222) the KM is 7.3 x 10-4 M. This means that at micromolar concentrations of dirt the modified enzyme will bind less dirt than the wild-type one, but the dirt concentrations caked onto our clothes are well above micromolar. The product turnover number, kcat (page 242), is 50 s-1 for subtilisin (met222) and 40 s-1 for subtilisin (ala222): The mutant enzyme is slightly slower, but not by much. By changing a met to an ala, a new enzyme has been produced that can do a reasonable job and is stable during storage and in our washing machines.

Green fluorescent protein is found naturally in certain jellyfish. Protein engineering has now created a palette of proteins with different colors (blue, cyan, green, and yellow). However, the great advantage of these proteins to biologists is that chimeric proteins (proteins composed of two parts, each derived from a different protein) incorporating a fluorescent protein are intrinsically fluorescent. This means that our protein of interest can be imaged inside a living cell using a fluorescence microscope (page 6). The fluorescent part of the chimeric protein tells us exactly where our protein is targeted in the cell and if this location changes in response to signals.

Figure 7.14 illustrates how this approach can be used to determine what concentration of glucocorticoid drug is required to cause the glucocorticoid receptor to move to the nucleus. The plasmid, like many plasmids designed for convenience of use, contains a multiple cloning site (MCS), sometimes called a polylinker, which is a stretch of DNA that contains several restriction endonuclease recognition sites. A convenient restriction endonuclease is used to cut the plasmid (in this case, for green fluorescent protein) and the cDNA for the glucocorticoid receptor is inserted. The plasmid also contains a promoter sequence, derived from a virus, that will drive the expression of the DNA into mRNA in mammalian cells that have been infected with the plasmid (or transfected). The chimeric protein, synthesized from the mRNA, will have one part coded for by the cDNA of interest—the other part being the fluorescent protein. The plasmid is grown up in bacteria and then used to transfect mammalian cells. In the absence of glucocorticoid the protein, and therefore the green fluorescence, is in the cytosol. When enough glucocorticoid is added, it binds to the chimeric protein, which then moves rapidly to the nucleus.

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