Provided are a TEV protease variant, a fusion protein thereof, a preparation method therefor and the use thereof. Provided are a TEV protease variant with unique properties obtained by screening and a fusion protein thereof. The TEV protease variant has a low enzyme cleavage activity during expression in hosts, and preferably has a lower enzyme cleavage activity compared to an S219V variant having an amino acid sequence as shown in SEQ ID NO: 10. The enzyme cleavage site of the TEV protease variant is selected from EXXYXQG/S/H, wherein X is any amino acid residue, and the enzyme cleavage site is preferably selected from SEQ ID NO: 7 and 8. Fusion expression using the TEV protease variant of the present invention and a polypeptide can be used for preparing a polypeptide quickly and efficiently, thereby solving the problems currently present in the process of the recombinant production of a polypeptide.

A biosensor comprises first and second molecular components and is capable of displaying protease activity in response to a binding event mediated by first and second binding partners of the biosensor. The first and second binding partners may bind each other directly or may both bind a target molecule. At least the first molecular component comprises an autoinhibited protease, whereby the binding event switches the protease frora an autoinhibited inactive state to a protease active state. The second molecular component may activate the protease of the first molecular component by binding a cross-binder which releases the autoinhibitor or by cleaving a linker which releases the autoinhibitor. The first and second molecular components may both have autoinhibited proteases which reciprocally activate each other.

The present invention relates to a method for expressing, water-solubilizing, and purifying protein by using a calsequestrin-tag (CSQ-tag). Provided are: a recombinant expression vector containing a CSQ-tag and a target protein; a host cell transformed using the recombinant expression vector; and a method for expressing and purifying a target protein by using a CSQ tag. The present invention uses CSQ-tags to increase the expression of proteins that are widely used in pharmaceuticals and cosmetics, and allows the proteins to be easily separated by calcium, and thus is expected to be able to lower the cost of protein materials and protein pharmaceuticals.

The present invention relates to a nucleic acid molecule encoding a fusion protein, wherein the nucleic acid molecule comprises: (a) a first nucleic acid sequence encoding a first biosensor, wherein said first biosensor is a first molecule capable of interacting with a second molecule; (b) a second nucleic acid sequence encoding an effector-activating module, wherein the effector-activating module comprises a nucleic acid sequence encoding a first part of a protease, wherein said first part of the protease is capable of interacting with a second part of said protease to form an active form of said protease; (c) a third nucleic acid sequence encoding a third biosensor comprising a protease cleavage site, wherein the protease cleavage site is sterically occluded in the absence of a stimulus for said third biosensor and wherein the protease cleavage site becomes accessible in the presence of said stimulus.

The present invention relates to a method for screening a protease variant and the obtained protease variant, and the protease variant has a special performance. The method for screening the protease variant comprises the following steps: (1) constructing a protease random mutation library, optimizing a protease random mutation virus library, and optimizing a protease random mutation phage library; (2) for the protease random mutation library, optimizing the protease random mutation virus library, and optimizing the protease random mutation phage library and screening a protease variant having weak protease enzyme-cutting activity in a host or a similar condition and having protease enzyme-cutting activity in an in vitro denaturation condition; and (3) the step of optionally characterizing the protease variant, wherein the protease optimizes TEV protease or enterokinase. The protease variant in the present invention facilitates industrial production of protein.

The present invention relates to a protein having proteolytic activity inducible and activable by the experimenter in the cytosol or in the secretory pathway, and uses thereof for controlling the maturation in a vital cell of a protein subject to proteolytic cleavage, and in a purification process of recombinant proteins.

Provided is a proteolysis-targeting virus, wherein one or more proteolysis-targeting molecules that can be recognized by the ubiquitin-proteasome system are comprised at one or more different sites of protein thereof, and the viral protein is linked to the proteolysis-targeting molecules by one or more linkers that can be selectively cleaved. Also provided are a nucleic acid molecule encoding the proteolysis-targeting virus, a nucleic acid vector expressing the proteolysis-targeting virus, a preparation method for the proteolysis-targeting virus, methods for the preparation of an attenuated live virus, replication-incompetentlive virus, replication-controllable live virus, and a relevant vaccine and medication for preventing and treating virus infections, a vaccine or pharmaceutical composition comprising the proteolysis-targeting virus, and a system for preparing the proteolysis-targeting virus.

It is intended to provide a fusion polypeptide that regulates the transcription of a target gene. The present inventors have provided a fusion polypeptide comprising: a cell-penetrating peptide; a DNA-binding polypeptide; and a transcriptional regulator.

Provided are fusion proteins and related molecules useful for conducting base editing with reduced or no off-target mutations. The fusion proteins may include a first fragment comprising a nucleobase deaminase or a catalytic domain thereof, a second fragment comprising a nucleobase deaminase inhibitory domain, and a protease cleavage site between the first fragment and the second fragment. Also provided are improved prime editing systems, including prime editing guide RNA with improved stability.

Provided are fusion proteins and related molecules useful for conducting base editing with reduced or no off-target mutations. The fusion proteins may include a first fragment comprising a nucleobase deaminase or a catalytic domain thereof, a second fragment comprising a nucleobase deaminase inhibitory domain, and a protease cleavage site between the first fragment and the second fragment. Also provided are improved prime editing systems, including prime editing guide RNA with improved stability.