Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Fundamentals of Molecular Biotechnology
Site-Directed Mutagenesis and Protein Engineering
Conclusion

The properties of any protein depend on its conformation, which in turn is determined by its Amino Acid Sequence. Certain Amino Acids in the polypeptide chain play a pivotal role in dictating the Specificity, thermostability, and other Functional Characteristics of a protein. Consequently, a single nucleotide substitution in a protein-coding Gene can lead to the incorporation of an amino acid that diminishes its activity or, conversely, enhances specific properties. With the advent of Recombinant DNA technology, it became possible to introduce precise alterations into cloned genes and engineer Proteins containing desired amino acids at specific sites—an approach known as Site-Directed Mutagenesis. Typically, the gene of interest is cloned into the DNA of phage M13. The single-stranded DNA form of this phage is replicated using an oligonucleotide primer synthesized to introduce a specific nucleotide into the target gene. Next, E. coli Cells are transformed with the double-stranded M13 DNA. A fraction of the resulting phage progeny carries the gene with the desired mutation. These particles are identified, the mutant gene is inserted into an expression vector, and the corresponding protein is synthesized and assayed for activity. Modifications to cloned genes can also be achieved using Plasmids or PCR. Since it is usually impossible to predict in advance exactly which amino acid(s) must be replaced to improve a particular property of a target protein, random mutagenesis is often preferred over oligonucleotide-directed mutagenesis.

The choice of which amino acid to replace is generally guided by its functional role within the protein, insights gained from genetic studies, or X-ray crystallographic data on the protein's three-dimensional Structure. By modifying specific sites or entire regions of a protein molecule, researchers can enhance thermostability, alter pH sensitivity, modify substrate specificity, adjust Allosteric Regulation, change cofactor requirements, and tune other properties. For instance, the thermostability of Triosephosphate isomerase was successfully increased by substituting amino acids at just two positions. This approach can be employed both to confer novel properties upon existing proteins and to design entirely unique Enzymes.

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Review Questions

1. What physical and Chemical properties of Enzymes can be altered using site-directed mutagenesis?

2. Suppose you have cloned a bacterial gene expressed in E. coli and wish to modify its activity. However, due to various technical reasons, standard M13 DNA mutagenesis yielded only a small fraction of clones with the mutant target gene, while the majority retained the intact gene. How can you increase the proportion of clones containing the desired mutant DNA?

3. Suppose you have isolated the gene for an enzyme synthesized in E. coli. Outline a strategy to alter the catalytic activity of this enzyme, given that you know The nucleotide sequence of the encoding gene, but do not know which region of the enzyme molecule is responsible for catalytic activity.

4. What are the Advantages and disadvantages of oligonucleotide-directed mutagenesis using M13 bacteriophage versus PCR?

5. Describe a strategy for oligonucleotide-directed mutagenesis using plasmid DNA.

6. How can degenerate primers be used to introduce random mutations into DNA?

7. Describe a strategy for increasing the stability of a protein that: a) lacks Cysteine residues; b) contains an odd number of cysteine residues.

8. How does replacing asparagine with another amino acid residue affect protein stability?

9. How can an enzyme's requirement for Cofactors be altered?

10. How would you go about modifying the catalytic activity or substrate specificity of an enzyme whose gene you have isolated? What is the rationale behind this procedure?