Split ubiquitin yeast two hybrid system
Ubiquitin can be divided into two parts: N-terminal (Nub) and C-terminal (Cub), with strong affinity between Nub and Cub. When expressed simultaneously in the same cell, Nub and Cub bind to form complete ubiquitin, which is recognized and cleaved by ubiquitin proteases (UBPs). After the mutation of isoleucine (I) in the third position of Nub into glycine (G), the affinity between Nub and Cub is greatly reduced. The bait protein is constructed on Cub, the Cub fusion transcription factor LexA-VP16, and the prey is constructed on NubG. When bait interacts with prey, Cub and NubG come close to form a complete ubiquitin, which is recognized and cleaved by UBPs. The cleaved transcription factor LexA-VP16 enters the nucleus and initiates the expression of reporter genes (His, Ade, LacZ).
Nano antibody library
Nano antibodies are single domain antibodies composed of variable regions of natural heavy chain antibodies that lack light chains in camel family animals (camels, alpacas, alpacas, and their close relatives). The skeleton sequence of nanoantibodies in the nanoantibody library comes from IGHV1S1 – IGHV1S1S5. By comparing 93 existing nanoantibody sequences in the PDB database, the design is as follows: point random mutation combinations were performed on CDR1-3 regions, with conserved sites and random sites with diversity of 2, 4, or 18. Among them, CDR3 region was designed with length polymorphism, with three lengths of 12, 16, and 20 amino acids, respectively.
Advantages and Application Scenarios
About one-third of the proteome is composed of membrane proteins, with a large number of membrane proteins having disease-related functions. Developing experimental methods for identifying membrane protein interactions is crucial for modern molecular biology. The split ubiquitin yeast two hybrid system can recognize the interactions between full-length integrated membrane proteins, membrane binding proteins, and soluble proteins on the cell membrane. Its advantage lies in its ability to cleverly solve the spatial contradiction between membrane protein interactions and nuclear reporter gene activation; Moreover, both Nub and Cub are small molecule peptides with only 40 amino acids, which will not cause spatial obstacles to the interaction of the target protein.
Most nanoantibodies come from the immunity of camels, which is a slow and expensive process that requires a large amount of purified proteins and large-scale animal husbandry and veterinary facilities. The nano antibody synthesis library can directly, quickly, and low-cost isolate nano antibodies.
Screening of NEU1 protein nanoantibodies using a split ubiquitin yeast two hybrid system