BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 2. PROTEIN ENGINEERING

Mutations and mutagenesis, and their application in protein engineering

Mutations are heritable alterations in The Genome Structure.

The process by which mutations arise is called mutagenesis.

Depending on the factors causing mutations, they are classified into Spontaneous and Induced. Spontaneous mutations occur randomly throughout an Organism's life under normal environmental conditions.

Induced mutations result from the exposure of an organism to Mutagenic Factors, primarily radiation and Chemical Mutagens.

Depending on the underlying molecular processes, mutations are classified into Gene, chromosomal, and genomic.

Genomic mutations involve A change in the chromosome number that is a multiple of the genome. For example, if the chromosome number in a diploid genome is designated as 2n, a genomic mutation can lead to The formation of polyploid organisms whose genomes contain 4n, 6n Chromosomes, etc.

Genomic mutation is also referred to as polyploidization. Depending on the Water/144.html">Origin of the chromosomes in polyploids, allopolyploidy and autopolyploidy are distinguished.

Allopolyploidy results from the combination of different genomes (Hybridization). Autopolyploidy is characterized by an increase in the chromosome number of a single genome that is a multiple of 2n.

Chromosomal mutations involve a change in the number (aneuploidy) or structure of individual chromosomes (Chromosomal aberrations).

The following types of chromosomal aberrations are distinguished:

- deletions — the loss of a portion of genetic material;

- duplications — the doubling of a portion of genetic material;

- inversions — a change in the orientation of chromosome segments;

- translocations — The transfer of a portion of genetic material from one chromosome to another.

Gene Mutations occur most frequently, involving structural changes within a single gene. Such mutations result in substitutions, deletions, insertions of one or more NUCLEOTIDES, or translocations, duplications, and inversions of various PARTS OF THE gene.

Mutations that alter only a single nucleotide are called point mutations. Point mutations are divided into two classes:

1) transitions — the replacement of a purine with a purine or a pyrimidine with a pyrimidine;

2) transversions — the replacement of a purine with a pyrimidine and vice versa.

There are 3 Genetic consequences of point mutations:

- preservation of the sense codon (synonymous nucleotide substitution);

- alteration of the sense codon, leading to the substitution of an amino acid at a specific position in the polypeptide chain (missense mutation);

- generation of a nonsense codon, resulting in premature termination (nonsense mutation).

Protein Engineering enables the creation and study of non-natural Proteins endowed with novel biochemical properties. The most common approach to generating new proteins and modifying existing natural ones is their Biosynthesis either in vivo or in vitro. Typically, the gene of the protein under study is first constructed, then cloned into a vector, and finally expressed.

To induce mutations in cloned genes for the purpose of obtaining mutant proteins, two main groups of Methods are used:

1) random mutagenesis (introducing multiple mutations at uncontrolled locations);

2) directed or site-specific mutagenesis (introducing mutations into precisely defined DNA regions).

Random mutagenesis is performed by incubating Nucleic Acids with chemical mutagens. This approach is widely applied in the directed Evolution of protein molecules.

Directed mutagenesis makes it possible to alter specific amino acid residues within protein molecules. It is most frequently employed in the rational design of proteins.

Mutations result in changes to the Amino Acid Sequence of the synthesized protein. By modifying specific sites of a protein molecule, one can enhance its thermostability, alter its pH sensitivity, Specificity, Allosteric Regulation, cofactor requirements, and other properties.