Protein engineering is the process of creating proteins that are useful or valuable in some way. It is a relatively new field, with much research being conducted into the understanding of protein folding and the recognition of protein design principles as well as other areas.
Rational protein design and directed evolution are two general strategies for protein engineering that can be used in conjunction with one another. These methods are not mutually exclusive; in fact, researchers will frequently use both at the same time. More detailed knowledge of protein structure and function, as well as advancements in high-throughput screening, have the potential to significantly expand the capabilities of protein engineering in the future. Even unnatural amino acids may eventually be included in the diet, thanks to newer methods such as the expanded genetic code, which allow for the encoding of novel amino acids in the genetic code.
Protein Engineering
To modify a protein sequence, researchers substitute, insert, or delete nucleotides in the encoding gene, with the goal of producing a modified protein that is more suitable for an application or purpose than the unmodified protein. Protein engineering is a branch of biotechnology that includes genetic engineering, biotechnology, and biotechnology-related fields. Protein engineering differs from the broader term “targeted mutagenesis” in that it focuses on specific applications. In targeted mutagenesis, also known as site-directed mutagenesis, an alteration is made to a specific site within a gene sequence. It is used in genetic research (Hutchison et al., 1978). Alterations of this nature can be carried out for engineering purposes, such as in protein engineering, or for the purpose of examining the effect of specific mutations in a gene.
Sexual methods
Homologous recombination carried out in vitro
The process of homologous recombination can take place either in vivo or in vitro, depending on the circumstances. In vitro homologous recombination replicates natural in vivo recombination. These methods of in vitro recombination need a high degree of sequence homology between the two sets of parental DNA.
These methods take advantage of the inherent diversity that is present in parental genes by recombining them in order to produce chimeric genes. The chimaera that was created has a mix of qualities inherited from both of its parents.
DNA shuffling
In the early days of recombination, one of the earliest procedures that was developed was an in vitro technique. It starts with the homologous parental genes being broken up into little pieces by DNase1, which is the first step. After that, the indigestible parental genes are separated from these smaller bits to be purified. After that, primer-less PCR is used to rejoin the fragments that were previously purified. Chimeric DNA is produced as a byproduct of this particular PCR, which involves the priming of each other by homologous fragments originating from distinct parental genes. After this step, end terminal primers are used in normal PCR to amplify the chimeric DNA of the parental size.
Random priming In vitro recombination (RPR)
This method of in vitro homologous recombination begins with the manufacture of a large number of short gene fragments using random sequence primers. Each of these fragments has a single point mutation. Using PCR that does not require primers, these fragments are pieced back together to form the full-length parental genes. After that, these reconstructed sequences are amplified by utilising PCR, and then they are put through additional selection processes. Because there is no use of DNase1 in this method, there is no bias for recombination occurring close to a pyrimidine nucleotide. This is an advantage over DNA shuffling, which is why this method is advantageous. This technique has a number of benefits, one of which is that it makes use of synthetic random primers that are of a consistent length and do not contain any biases. Last but not least, the length of the DNA template sequence is irrelevant to the success of this approach, which only needs a small quantity of the parent’s DNA.
Truncated Metagenomic Gene-Specific PCR
This technique creates chimeric genes by starting with metagenomic materials and working backwards. The first step involves isolating the gene of interest from a sample of metagenomic DNA via functional screening. Following this step, special primers are constructed and then utilised in order to amplify the homologous genes that have been extracted from the various environmental samples. Finally, chimeric libraries are formed by shuffling these amplified homologous genes in order to obtain the desired functional clones. Chimeric libraries are constructed in order to retrieve the desired functional clones.
Asexual Methods
The use of asexual reproduction does not result in the formation of any cross connections between the genes of the parents. Using a variety of mutagenesis approaches, individual genes are what are needed to generate mutant libraries. The mutagenesis that results from these asexual procedures can be either random or targeted.
Embedded Arrays for Mutagenesis (TaGTEAM)
This technique has been applied in yeast in order to generate targeted in vivo mutations. In this particular technique, a 3-methyladenine DNA glycosylase is joined to a tetR DNA-binding domain.
It has been demonstrated that this results in an increase in mutation rates that is more than 800 times greater in sections of the genome that include tetO sites.
Mutagenesis by Random Insertion and Deletion
This technique includes simultaneously deleting and inserting chunks of bases of any length in order to bring about the desired change in the sequence’s overall length. It has been demonstrated that using this technology can result in the production of proteins with new capabilities. This is accomplished through the introduction of new restriction sites, particular codons, and four base codons for non-natural amino acids.
Directed Evolution
The processes of sequence diversification and screening are frequently repeated multiple times in directed evolution, with additional amino acid substitutions accruing in each round and each round providing a protein sequence that is closer to that of the protein engineering target. To carry out sequence diversification, directed evolution employs techniques from molecular biology; to screen the resulting proteins for desired properties, it uses techniques from biochemistry, analytical chemistry, and microbiology (among other fields).
Protein engineering efforts have primarily focused on improving the efficacy and pharmacokinetic profiles of recombinant proteins by altering the structural characteristics of the proteins. If we consider the drug insulin, it has been discovered that upon storage of a drug product composed of individual native insulin molecules, dimeric and hexameric insulin structures form. It was discovered that higher order species had a longer residence time at the site of injection (either subcutaneously or intramuscularly) in humans, and that this resulted in an inordinately delayed onset of the therapeutic effect. In order to overcome this difficulty, researchers altered the amino acid sequence in the regions of the protein associated with the propensity for self-association in order to reduce aggregation and generate insulins that were more rapidly acting.
The protein engineering of insulin has also been pursued in order to increase the duration of the hormone’s action in the human body. Several modifications were made to Levemir® to allow for reversible binding to albumin both at the site of injection and later in the plasma. This allows for a prolonged release of insulin as well as an increased duration of action of the drug.
The following are some examples of biotechnology engineering:
- Agricultural biotechnology
- Environmental biotechnology
- Industrial biotechnology
- Genomics
- Hybridoma technology
- DNA Cloning
- Health technology
Conclusion
Protein engineering is an area of recombinant DNA technology that has experienced rapid growth in recent years. Managements in genes are expressed as changes in protein conformation that are responsible for the desired properties of the protein. In general, a variety of techniques for the specific engineering of proteins can be divided into two categories: techniques that necessitate extensive prior knowledge of the protein and techniques that help to establish the concept of rational technique of directed evolution that aids in the expression of the progression of natural evolution. Since its inception, protein engineering has thrived in order to produce proteins that have a variety of rewarding applications in industries such as industry, health and medicinal sciences, and ultimately in nanobiotechnology, which is currently the case.