Positional mutation is a protein engineering technique that substitutes, inserts or deletes specific nucleotides in known DNA sequences based on the known structure and function of proteins to produce mutant protein (enzyme) molecules with novel traits. The technology is widely used in the biological and medical fields. Position mutation technology has the characteristics of high mutation rate, simple and easy to perform, and good repeatability. As a research method, localization mutation technology is also widely used to study the relationship between protein structure and function, so as to elucidate the regulation mechanism of genes, the etiology and mechanism of diseases.
The “small change” of protein molecules based on natural protein structure refers to the modification, substitution or deletion of a few residues of proteins of known structure. This is the most widely used method in protein engineering, and can be mainly divided into proteins. Two types of modification and gene location mutation. Gene-localized mutation refers to the transformation of protein molecules at the genetic level, that is, the method of site-directed mutagenesis, the insertion, deletion, substitution and reorganization of nucleotide codons of genes encoding proteins, and then the mutated genes are carried out. The protein expresses and analyzes the functional activity of the expressed protein, and the result provides a new design for protein molecular engineering.
Design goals and solutions for location mutation
The common design goals of localization mutations are to improve the heat and acid stability of proteins, increase activity, reduce side effects, improve specificity, and conduct structural-functional studies through protein engineering. Hartley is equal to 1986 to complete a design goal and solution that we want, and still has important reference value. The stability of protein is an important prerequisite for the normal biological activity of proteins. Therefore, improving the stability of proteins has become one of the important goals of protein design and transformation.
Type of mutation
There are many ways to change the nucleotide sequence of a gene, such as chemical synthesis of genes, direct modification of genes, and cassette mutation technology. Depending on the manner in which the gene is mutated, it can also be classified into three categories: insertion of one or more amino acid residues; deletion of one or more amino acid residues; replacement or substitution of one or more amino acid residues. In order to achieve the purpose of gene location mutation, in vitro recombinant DNA technology or PCR method is often used.
The amino acids in a protein are determined by the triplet codon in the gene. By changing one or two bases, the amino acid species can be changed to produce a new protein. It is usually the amino acid that changes a position in the functional region to study the structure, stability or catalytic properties of the protein. The work of point mutation is the main body of current protein engineering research. So far, many kinds of proteins such as subtilisin, T4 lysozyme, dihydrofolate reductase, trypsin and ribonuclease have been modified. For example, replacing Asn117 of a tissue-type plasminogen activator (t-PA) with Glu117, thereby removing an original glycosylation site; since the original sugar chain can promote t -PA is cleared from plasma, so point mutations can reduce plasma clearance of t-PA and prolong plasma half-life.
In 1985, Wells proposed a genetic modification technique for a box-type mutation that can produce 20 different amino acid mutants at one site, and can perform “saturation” analysis of important amino acids in protein molecules. Using the localization mutation, two original vectors and endonuclease cleavage points not present on the gene are added on both sides of the amino acid code to be modified, and the endonuclease is used to digest the gene, and then the synthesized double-stranded DNA fragment with different changes is substituted for digestion. part. A variety of mutant genes can be obtained in such a single treatment.
Procedure for locating mutations
The protein molecular design program for gene localization mutation follows the procedure in the design principle, but the gene location mutation has its own particularity, and its specific procedure is as follows.
- Establish a structural model of the protein under study
Establishing a three-dimensional structural model of a protein is critical to establishing a mutation site or region and predicting the structure and function of the mutated protein. The structure can be determined by X-ray crystallography, two-dimensional nuclear magnetic resonance, or the like, or a structural model can be established based on the structure of the analog or other structural prediction methods.
- Identify locations that have a significant impact on the required properties
- Predict the structure of the mutant
- Construct mutants. Mutant protein
- Examination of mutant proteins
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