Automatic protein expression quantitative analysis system

The establishment of the two-hybrid system is due to the understanding of the transcription initiation process regulated by eukaryotes. Initiation of cell gene transcription requires the participation of trans-transcription activators.

Studies in the 1980s showed that transcription activators are modular in structure, that is, these factors are often composed of two or more independent domains, including DNA binding domain (referred to as DB) and transcription activation domain (AD), they are necessary for the function of transcription activator.

Although the DB alone can bind to the promoter, it cannot activate transcription. The hybrid proteins formed by different transcription activators DB and AD still have the normal function of activating transcription. For example, the Gal4 protein DB of yeast cells and an acidic activation domain B42 of E. coli can still bind to the Gal4 binding site and activate transcription.

The work of Fields et al. marks the official establishment of the two-hybrid system. They modeled the two proteins Snf1 and Snf2 related to the regulation of the SUC2 gene, fused the former to the Gal4 DB domain, and the other to the acidic region of the Gal4 AD domain.
The fusion protein formed by DB and AD is now generally called "bait” and "prey" or target protein. If there is an interaction between Snf1 and Snf2, then DB and AD located on these two fusion proteins can reform the active transcription activator, thereby activating the transcription and expression of the corresponding gene. This activated gene that shows the interaction between "bait" and "prey" is called a reporter gene.

By detecting the expression product of the reporter gene, in turn, it can be determined whether there is an interaction between the two proteins used as "bait" and "prey". Then Fields et al. used LacZ encoding β-galactosidase as a reporter gene, and introduced the GAL1 sequence regulated by Gal4 protein in the upstream regulatory region of the gene.

This modified LacZ gene was integrated into the yeast chromosome URA3. Yeast's GAL4 gene and GAL80 gene (Gal80 is a negative regulator of Gal4) are deleted, thus eliminating the influence of endogenous regulators in the cell. It is known that there is an interaction between Snf1 and Snf2. As a result, it was found that only yeast cells transformed with both Snf1 and Snf2 fusion expression vectors had β-galactosidase activity, and any of these vectors alone could not detect β-galactosidase activity.

Most of the two-hybrid systems currently developed are based on the systems established by Fields et al. These new systems have mainly made improvements to reporter genes, "bait" expression vectors, and "prey" expression vectors.

One of the important improvements is the introduction of additional reporter genes, such as the widely used HIS3 gene. After transforming yeast cells with the HIS3 reporter gene, only when HIS3 is started to express can it grow on selective media lacking histidine. The transcriptional expression of the HIS3 reporter gene is initiated by the interaction of "bait" and "prey".

Most two-hybrid systems often use two or even three reporter genes simultaneously, one of which is LacZ. These modified genes have the same transcription activator binding site in the promoter region, so they can be activated by the same transcription activator (such as the Gal4 protein described above). This double or multiple selection not only improves the detection sensitivity but also reduces the false positive phenomenon. Other improvements include "bait" or "prey" expression vectors.

In the two-hybrid identification process, two transformations are required. This workload is quite large, especially when looking for new proteins to act on. Moreover, the transformation efficiency of yeast cells is about 4 orders of magnitude lower than that of bacteria. Therefore, the transformation step becomes the bottleneck of the two-hybrid technology. Bendixen et al. used yeast zygosity to avoid two transformation operations, while at the same time improving the efficiency of two-hybrid.

Two types of coordination are involved in the sexual reproduction of yeast: a conjugated type and alpha conjugated type. The mating between these two haplotypes can form diploid, but a conjugated type cell or alpha junction cannot join together to form diploid.
According to this characteristic of yeast sexual reproduction, they transformed the library plasmid into α-zygote yeast cells, and the “bait” expression vector into a-zygote cells.

Then spread the screening plates separately to make the cells grow into lawns, and then copy the two kinds of lawns to the same triple screening plate. In principle, only diploid cells that interact with the bait and the target protein can grow up on this plate. Haploid cells or those that are diploid but the DB fusion protein and AD fusion protein do not interact are eliminated.

The grown clones were further identified by β-galactosidase activity. This improvement not only simplifies the experimental operation, but also improves the screening efficiency of two-hybrid.

In the process of studying the structural and functional characteristics of proteins and the mode of action, sometimes the interaction between proteins must be destroyed by means of mutations and the addition of inhibitors. In response to this need in practical work, Vidal et al. developed the so-called reverse two-hybrid system. The key to this technology is the introduction of the reporter gene URA3.

The URA3 gene plays a counter-selective role here, and the enzyme it encodes is the key enzyme for uracil synthesis. This enzyme converts 5-fluoroorotic acid (5-FOA) into substances that are toxic to cells. Vidal et al. introduced Gal4 binding sites in the promoter of the URA3 gene by modification.

This modified yeast strain can grow only when the interaction between "bait" and "prey" activates the expression of the URA3 gene on selective media lacking uracil. The interaction of "bait" and "prey" on the complete medium containing 5-FOA inhibits cell growth.
However, if the protein of interest, that is, the protein fused to DB or AD is mutated or no longer interacts due to the interference of external drugs, and the URA3 gene is not expressed, the cells can grow on complete medium containing 5-FOA.

In this way, Vidal et al. screened for mutations in the transcription factor E2F1. These mutants still bind to the retinoblastoma protein RB, but lose the ability to bind to another protein called DP1. The results were verified by in vitro binding experiments. By sequencing these mutant protein genes, they discovered a new site where E2F1 binds to DP1.

The yeast two-hybrid system described above is based on the activation of RNA polymerase II.

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