What is protein mutagenesis

  • Protein mutagenesis refers to the introduction of desired changes (usually in a favorable direction), including addition, deletion, and point mutations, into the target DNA fragment (either genome or plasmid) by means of polymerase chain reaction (PCR) and other methods. Site-directed mutation can rapidly and efficiently improve the character and characterization of the target protein expressed in DNA, which is a very useful method in gene research.

    Site-directed mutagenesis in vitro is an important experimental method in current biological and medical researches. It is a convenient program to modify and optimize genes, an effective means to explore promoter regulatory sites, and a powerful tool to study the complex relationship between protein structure and function.

    The structure of protein determines its function, and the relationship between them is one of the focuses of proteome research. Fixed-point modification, deletion or insertion of a specific base of a known gene can change the corresponding amino acid sequence and protein structure, and the study on the expression products of the mutant gene can help human understand the relationship between protein structure and function, and discuss the structure/domain of protein. And using the fixed point mutation gene technology transform: such as the wild type of green fluorescent protein (wtGFP) is to a faint green fluorescent under uv excitation, after the light emitting structure domain specific amino acids of fixed-point modification, GFP can be excitation in the visible light wavelength range (absorption area red shift), and one hundred times on the luminous intensity is better than the original, and even appeared yellow fluorescent protein, blue fluorescent protein, and so on. Fixed point mutation technology potential application field is very wide, such as the research of the structure of protein interaction sites, different modification enzyme activity or dynamic characteristics, modification or DNA promoter action element, the introduction of new enzyme loci, improve the antigenicity of protein or protein stability, activity, the research of crystal structure, as well as drug development and gene therapy, etc.

    Differs from that of fixed point mutation is the random mutation based on PCR, by changing the PCR conditions improve the random error in the process of PCR, this method is suitable for the unknown protein, a global analysis, so someone called saturated mutations (saturation mutagenesis). However, this method has limitations in application due to its lack of purposiveness, tedious analysis and absence of more than two base mutations at one site. Site-directed mutagenesis is applicable to genes with a preliminary understanding of protein structure, which is more purposeful, precise and simple than random PCR mutagenesis. Meanwhile, gene modification is more "arbitrary". As site-directed mutation technology has a very broad application prospect in proteomics, it is believed that this technology will have greater improvement and development in the near future, and it will also be familiar to more people.

    According to the theoretical knowledge of biology, genes will have mutations, which can be spontaneous or induced. But before the invention of site-directed mutagenesis by Canadian biochemist M Smith (1932-2000), mutant strains had to be induced to mutate by natural or chemical means. Such a method is a random mutation, and the mutant strain must be changed in biological shape to be sure that there is a mutation, but the mutation location cannot be determined unless it is found by molecular biological method or genetic method. In other words, the mutation is blind. But Smith's site-directed mutagenesis, which allows random or engineered mutations to be carried out on any gene fragment via a designed oligonucleotide, is so targeted that it is sometimes called "anti-inheritance".

    It's interesting that this revolutionary approach to life science research and application was the result of a coffee break between Smith and his colleagues. Almost every biological laboratory USES site-directed mutagenesis to study the function of genes or proteins. The application of site-directed mutation method is not only widely used in the field of genetic engineering technology, but also can be used in the agricultural breeding of good varieties resistant to insects and diseases, as well as the medical correction of genetic diseases and the treatment of cancer and other diseases.