Protein engineering has revolutionized the development of enzymes for use in biosensors and bioelectronics by allowing for directed changes to enhance or alter enzyme function. Here are the key aspects of this field:
Approaches to Protein Engineering
There are two main approaches to engineering enzymes:
Rational Design
- This approach involves predicting the necessary changes to achieve a specific alteration in function. It requires:
- The cloned gene for the protein of interest.
- X-ray structure or detailed structural information.
- Low-throughput screening or selection methods.
- Modest to high computational needs.
- Mutations are typically introduced using mutagenic primers[6].
Evolutionary Design
- This approach involves making random changes and then selecting or screening for the desired properties. It requires:
- The cloned gene for the protein of interest.
- No need for X-ray structure.
- High-throughput screening of large mutant libraries.
- Error-prone PCR, DNA shuffling, or mutator strains to introduce mutations.
- Low to modest computational needs[6].
Methods for Mutagenesis and Expression
- Mutagenesis: This can be achieved through various in vitro methods, nearly all of which involve the Polymerase Chain Reaction (PCR). Techniques include site-specific changes, random mutations using error-prone PCR, DNA shuffling, and mutator strains[6].
- Expression: The modified DNA is introduced into an expression host, and the modified protein is produced. This workflow from DNA to protein involves several steps, including mutagenesis, vector construction, and expression in a host organism (Figure 15)[6].
Types of DNA Sequence Changes
- Base Changes: Resulting in amino acid changes or point mutations.
- Deletions: Removal of bases.
- Tagging: Introducing additional sequences at the ends of the protein.
- Loop Insertions: Introducing additional sequences within the protein.
- Protein Fusions: Joining two genes together to create a single polypeptide chain with multiple active sites or functionalities[6].
Modular Design Approach
Protein engineering often adopts a modular design approach, where the final protein is considered to be comprised of several functional modules:
- Signaling Module: Responsible for communicating externally via electronic or photonic exchange with a device.
- Active Site Module: Carries out the enzymatic function, typically catalysis.
- Immobilization Module: Involved in immobilizing the protein on a surface. This can include fusion proteins with specific tags that interact with the surface material (Table 3)[6].
