Protein Engineering

Protein engineering is the process of developing useful or valuable proteins. It is the new branch of biotechnology which understands protein folding and recognition for design principles. It is the second generation of recombinant DNA technology. It involves altering cloned DNA in vitro by novel mutational technique so that translated proteins have slightly altered properties. Protein engineering is the merging of several disciplines like molecular biology, protein chemistry, enzymology, structural chemistry to alter catalytic or structural stability of protein. With the development in genetic engineering, genes can be isolated from organism and for the synthesis of naturally occurring proteins.

Table of Contents

Objectives of protein engineering

Increasing substrate affinity to enzyme
Makes the enzyme heat tolerant (active at high temperature) and pH stable.
Enhances the substrate specificity by modifying the substrate binding site of the enzyme.
Designing the enzyme to make it resistant to proteolytic degradation.
Synthesizing enzyme that is stable and active in non-aqueous solvents.
Changing the enzyme in order to make it independent of cofactor for its function.
Improving the stability of enzyme to heavy metals.
Fusing the enzymes needed in the reactions to give a final product.
Produce hybrid enzymes.
Make isolation and purification of enzymes simpler.

Techniques used for protein engineering

There are two general strategies for protein engineering, one is genetic modifications (mutation) and another is chemical modifications.

Genetic modifications (mutation)

Mutation is defined as a change in the nucleic sequence (bases) of an organism’s genetic material. In simple words, mutations are accidental changes in a genomic sequence of DNA. It involves large sections of DNA becoming duplicated, usually through genetic recombination. There are two subtypes of genetic modifications, one is site directed mutagenesis and another is localized random mutagenesis.

Site directed mutagenesis

The change in nucleic acid sequence (or genetic material) of an organism at a specific predetermined location is known as the site directed mutagenesis. Using this approach site specific change (mutagenesis) can be made in the gene to produce desired enzyme. Directed mutagenesis can be done using M13 plasmid DNA, PCR, random primers, degenerative primers, nucleotide analogues, etc.

Methods for site directed mutagenesis

The single primer method

In this method of mutagenesis, the primer is a chemically synthesized oligonucleotide (7-20 nucleotides long). The starting material is single stranded DNA (to be mutated) carried in an M13 phage vector. On mixing this DNA with primer, the oligonucleotide hybridises with the complementary sequences, except at the point of mismatched nucleotide. Hybridisation is possible by mixing at low temperature with excess of primer and in the presence of high salt concentration. There are three types of variations in the oligonucleotide directed mutagenesis,

Multiple point mutagenesis
Insertion mutagenesis
Deletion mutagenesis

Cassette (small DNA fragment) mutagenesis

In cassette mutagenesis a synthetic double stranded oligonucleotide containing desired mutant sequence is used. Cassette mutagenesis is possible if the fragment of gene to be mutated lies between two restriction enzyme cleavage sites. The use of cassette mutagenesis allows total control over the type of mutation that can be generated.

Chemical modifications

In the chemical modification method, functional group on site chain of natural enzyme can be changed. Total chemical synthesis of an enzyme is only a theoretical possibility. Using this technique attachment of coenzyme to enzyme is possible. It is applicable to amino acids that have reactive side chains and only specific alterations can be made. Modification of single amino acid is difficult.

Applications of protein engineering

Investigative tools: Specific mutations in DNA allow the function and properties of DNA sequence in a rational approach.
Commercial applications: Broad range of enzymes are been used in different industries like, food, paper, leather, cosmetic, chemical and pharmaceutical industry. Food industry uses variety of enzymes like proteases, lipases, amylases, etc in food processing. These processes require high temperature, different pH and many other compounds for enzyme activity. To overcome these problems and to further enhance production and activity, properties of enzymes are modified using protein engineering.
Environmental application: Oxygenases, laccases and peroxidases are three major classes of enzyme which have significant role in environmental application for biodegradation of organic and toxic pollutants. Using protein engineering these enzymes are protected from enzyme denaturation by toxic compound and their catalytic activity is increased.
Medical and clinical applications: Protein engineering is useful in production of secreted therapeutic proteins like interferons, insulin, etc. Development of therapeutics against cancer is the major field of interest in protein engineering. Advancements in recombinant DNA technology and protein engineering enable the synthesis of novel antibodies which can be used as anti-cancer drug. Functional protein and peptides are engineered offering an efficient vehicle for adequate and targeted delivery of drug.

Summary

Protein engineering involves the modification and design of proteins to enhance their functionality or create novel structures. Techniques like site-directed mutagenesis and recombinant DNA technology enable the manipulation of amino acid sequences, leading proteins with improved properties. This field has diverse applications from developing therapeutic proteins to designing enzymes for industrial processes. Protein engineering plays a crucial role in advancing biotechnology and addressing various challenges in medicine, agriculture and many more.