Yeast two-hybrid technology, as the main method of studying protein interaction, occupies a pivotal position in large-scale protein interaction research, and has become an important experimental method in the field of molecular biology research. Therefore, many biological companies provide everything from constructing hybrid libraries to One-stop yeast two-hybrid service for library screening. However, the molecular biology operation of yeast two-hybrid technology is more complicated, including the construction of gene vector, protein self-activation detection, and confirmation of multiple yeast transformations and interactions. Improving yeast two-hybrid technology can improve its work. efficient.
The yeast two-hybrid service currently provided on the market includes nuclear system and membrane system. Compared with other interactive screening methods, the biggest advantage of yeast two-hybrid is that it can adapt to high-throughput screening at the genome level. Medicilon provides yeast two-hybrid service. It has independent high-quality laboratories and professional experimenters. It provides detailed yeast two-hybrid experiment procedures, original data and pictures, result analysis, and can issue authoritative test data reports.
In order to reduce the workload and make it more suitable for high-throughput screening, the yeast two-hybrid technology needs to be improved. The methods often used include yeast two-hybrid array screening technology and large-scale vector construction methods.
The yeast two-hybrid library screening technology developed from the traditional yeast two-hybrid technology can support the screening of a bait and the entire cDNA library, and can be used to screen the interaction between a protein and the entire cDNA library. However, this technology is not suitable for interaction screening at the genome level.
First, genomic-level interaction screening requires thousands of baits, and library screening of these baits is a huge workload; secondly, due to different cDNA fragments, strains that are at a disadvantage during the screening process are not easy to be screened; and finally screening. The library also faces a single screening and only a very small part of the interaction results in the library can be obtained. In order to adapt to the interaction screening at the genome level, yeast two-hybrid array screening technology has been developed.
The array screening method can be divided into one-to-one array, high-throughput array method, and sub-library-to-sublibrary method. The one-to-one array method screens strains containing different BD-X and all strains containing different AD-Y in a one-to-one correspondence.
The high-throughput array method is a screening method that combines all the different ORF-ADs into a library and then interacts with the different BD-Xs arranged in an array. Its essence is the screening of arrayed libraries in the case of a large number of decoys. The sub-library-to-sub-library method numbers the members of the larger total number of DBD-X and AD-Y respectively and group them, each group contains a certain number of members, and then the members of each group are assembled to form a pool, and then the prey sub-pool is used Act on the decoy sub-pool one by one to screen positive clones. This method requires a lot of work, and both the prey and bait of the positive clones obtained need to be sequenced and identified.
Large-scale yeast two-hybrid screening requires the construction of a large number of expression vectors. Therefore, the quality and speed of vector construction work is very important for subsequent yeast two-hybrid screening. The traditional method of constructing a vector is restriction digestion-ligation method, which can effectively insert the target fragment into the vector. However, its operation time is long and requires a two-step reaction of digestion and ligation. The successfully constructed recombinant plasmid needs to be verified by DNA sequencing For its correctness, there are shortcomings such as vector self-connection and difficulty in the construction of expression vectors. These shortcomings make the restriction enzyme-ligation method not suitable for high-throughput vector construction.
Gene cloning is a basic operation method in molecular biology. The construction of vectors through double enzyme digestion is a commonly used method. This method uses the principle that restriction enzymes can recognize and cleave specific nucleotides. Use two different restriction endonucleases to cut the target gene to obtain the target gene fragment with sticky ends before and after, and connect it with the linear vector with the same sticky end that is also cut by two restriction enzymes in T4DNA The ligation is carried out under the action of enzymes, so as to realize the problems of gene cloning efficiency and low ligation efficiency, which makes it quite difficult for the target gene fragment to be inserted into the vector. For this reason, people have adopted a variety of strategies for cloning. Later, a gene cloning method called homologous recombination was developed.
In contrast, the homologous recombination method is relatively simple and easy to construct a vector. Compared with the double restriction digestion vector construction method, the construction process of this method is somewhat different. First, the target gene fragment amplification primers are different from conventional primer design; secondly, only a single enzyme digestion is required during the vector cutting process; third, the connection between the vector and the target gene fragment is based on homologous recombination rather than sticky end complementation. In the actual operation process, the double enzyme digestion method and the homologous recombination method have their own advantages. The most representative method of homologous recombination is the Gateway method.
Gateway utilizes site-specific recombination, so after constructing the entry vector, restriction endonucleases and ligases are no longer required. Once you have an entry clone, you can use it multiple times to transfer the target gene to various expression vectors (target vectors) modified by Gateway. In addition, since the reading frame and orientation of the DNA fragments remain unchanged during recombination, there is no need to worry about sequencing new expression clones. When using each new expression system, more time will be saved.
Gateway technology is a novel universal system for cloning and subcloning DNA sequences, which facilitates the analysis of functional genes and the expression of proteins. Once in this versatile operating system, DNA fragments can be transferred between vectors through site-specific recombination.
Gateway technology is based on the well-researched lambda phage site-specific recombination system (attB x attP →attL x attR). The two reactions of BP and LR constitute the Gateway technology. The BP reaction uses a recombination reaction between an attB DNA fragment or expression clone and an attP donor vector to create an entry clone. The LR reaction is a recombination reaction between an attL entry clone and an attR target vector. The LR reaction is used to transfer the target sequence to one or more target vectors in parallel reactions.
In the BP reaction, gene transfer forms the entry clone, and in the LR reaction, the entry clone can be used as a reaction product to generate the final expression clone. It only takes two steps to complete the construction of the Gateway expression clone:
(1) Create an entry clone and clone the target gene into the gate vector by PCR or traditional cloning methods.
(2) Mix the entry clone containing the target gene with the appropriate target vector and Gateway LR Clonase enzyme to construct an expression clone. (Expression cloning is used for protein expression and analysis in a suitable host.)
Yeast two-hybrid technology is simple, sensitive, efficient, and can reflect the interaction between different proteins in living cells. The improvement of its technology will make it more widely used in the research of protein interaction. .
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