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 molecule comprises: a protease amino acid sequence; at least one sensor comprising at least one sensor amino acid sequence which is responsive to at least one target molecule; and an inhibitor of the protease activity of said protease amino acid sequence; wherein the biosensor is switchable from a protease active to a protease inactive state, or from a protease inactive to a protease active state when said sensor responds to said target molecule. The biosensor protease may be a protease of a virus such as a Potyvirus or a Flavivirus wherein the inhibitor is an autoinhibitory peptide derived from the virus. The biosensor may respond to the target molecule allosterically or may be cleaved by a target protease molecule.

The present invention relates to enzyme inhibitors. More specifically, the present invention relates to ligand-directed covalent modification of proteins; method of designing same; pharmaceutical formulation of same; and method of use.

Disclosed herein include methods, compositions, and systems suitable for use in delivering a polynucleotide to a target cell of a subject in need thereof. In some embodiments, a viral vector comprises a polynucleotide encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), RNA-dependent RNA polymerase (L), and one or more transgenes. The viral vector can comprise one or more of a conditionally stable fusion protein, a protease fusion protein, a degron fusion protein, and/or a glycoprotein derived of another species than the viral vector polynucleotide to enable control of viral vector transduction and/or replication.

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 disclosure is directed to an isolated polynucleotide encoding a modified picornavirus 3C protease, wherein the modified picornavirus 3C protease includes an altered secondary structure and one or more amino acid substitution(s) located at one or more amino acid position(s) corresponding to positions 16-25, 99-100 and 115-130 of a wild-type Fool-and-Mouth Disease Virus (FMDV) 3C protease, wherein the isolated polynucleotide encoding the modified picornavirus 3C protease, when transformed into and co-expressed in a host cell, enhances transgene expression of a P1 precursor polypeptide in comparison to an amount of P1 precursor polypeptide transgene expression exhibited in a host cell transformed and co-expressed with a control picornavirus 3C protease, wherein the one or more corresponding amino acid position(s) is/are identified by an alignment of the modified picornavirus 3C protease with the one or more of the wild type FMDV 3C protease(s). Methods for processing a picornavirus P1 precursor polypeptide into picornavirus viral proteins and/or virus-like particles using the isolated polynucleotides are also provided.

The present disclosure provides danoprevir/NS3a complex reader (DNCR) and grazoprevir/NS3a complex readers (GNCR) polypeptides, fusion proteins, and combinations and their use.

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 invention relates to genetic incorporation of 2,3-diamino propionic acid (DAP) into polypeptides, to unnatural amino acids comprising DAP, to a tRNA synthetase for charging tRNA with unnatural amino acids comprising DAP, and to methods of using the resulting polypeptides, for example in capturing substrates and/or intermediates in enzymatic reactions. The invention also relates to compounds of formula (I) or (II): or salts, solvates, tautomers, isomers or mixtures thereof.

##STR00001##

Compounds having drug and bio-affecting properties, their pharmaceutical compositions and methods of use are set forth. In particular, betulinic acid derivatives that possess unique antiviral activity are provided as HIV maturation inhibitors, as represented by compounds of Formula I:

##STR00001##

These compounds are useful for the treatment of HIV and AIDS.