Engineered fusion proteins comprising a self-excising degron for controlling protein production are disclosed. In particular, the inventors have constructed fusion proteins comprising a degron connected to a protein of interest through a cleavable linker comprising a hepatitis C virus (HCV) protease site. The degron can be removed from the protein of interest by a czs-encoded HCV protease such that the protein of interest can be produced with minimal structural modification. Clinically available HCV protease inhibitors can be used to block protease cleavage such that the degron is retained after inhibitor addition on subsequently synthesized protein copies. The degron when attached causes rapid degradation of the linked protein. Such fusions of a degron to a protein of interest will be especially useful when control over protein production with minimal structural modification is desired.

Described herein are flavivirus virus-like particles (VLPs) that display on their surfaces antigenic flavivirus proteins. Also described are methods of making and using these VLPs.

The present invention provides systems and methods for performing forward genetic screens in cells and methods of treating disease through inhibiting targets identified in a forward genetic screen as involved in a pathway associated with the disease. The invention involves the use of multiple tandem fluorescent reporter molecules separated by cleavage sites under the control of a single promoter to enhance the fluorescent readout from a cell screen.

In alternative embodiments, provided are compositions, e.g., multiplexed platforms or systems, to screen for small molecule drugs that inhibit enzymes such as proteases, e.g., viral proteases, including Zika Virus (ZIKV). In one embodiment, provided are cell-based or multiplexed platforms for monitoring the activity of enzymes, e.g., proteases such as Zika Virus (ZIKV) viral proteases. In alternative embodiments, provided are assays that monitor the activity of the viral ZIKV NS3 protease, as observed by the cleavage of its boundaries with its cofactor NS2B and its downstream nonstructural protein NS4A.

The present specification discloses expression constructs comprising single-chain proteins comprising a di-chain loop region comprising an exogenous protease cleavage site and a protease that can cleave the exogenous protease cleavage site located within the di-chain loop, cell compositions comprising such expression construct, and intracellular methods of converting the single-chain protein into its di-chain form.

Provided are methods of selecting for cells that comprise two or more separate expression constructs. In certain embodiments, the methods comprise contacting a population of cells with two or more separate expression constructs under conditions in which the two or more expression constructs are delivered to cells of the population of cells. The two or more separate expression constructs comprise a first expression construct that encodes a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. The second expression construct encodes a protein required for cell surface expression of the selection marker. Such methods further comprise selecting for cells exhibiting cell surface expression of the selection marker. Related cells, compositions, kits and therapeutic methods are also provided.

The subject technology relates generally to compositions and methods for producing plasmid DNA of a desired quality. In addition, it relates to the discovery of Escherichia coli (E. coli) bacteria with a constitutive methylase gene stably incorporated into the chromosomal DNA and uses thereof.

This application is directed generally to foot-and-mouth disease virus (FMDV) 3C proteases that have been modified by mutating a polynucleotide sequence coding for the FMDV 3C protease. The modified FMDV proteases exhibit proteolytic activity on FMDV P1 precursor protein and exhibit a reduction in one or more toxic or inhibitory properties associated with an unmodified FMDV 3C protease on a host cell used to recombinantly produce it. Vectors carrying polynucleotides encoding modified FMDV 3C protease sequences can induce production of FMDV virus-like particles in a host cell when expressed in the host cell. The modified FMDV 3C proteases can generally be used to produce immunogenic FMDV preparations capable of inducing an immune response against FMDV.

Disclosed is a method for producing a recombinant protein of interest, characterised in by the following steps: (a) providing a fusion protein comprising an N.sup.pro autoprotease moiety and a protein of interest moiety in inclusion bodies, (b) solubilising the fusion protein in the inclusion bodies by subjecting the inclusion bodies to a solubilisation buffer containing a detergent and wherein the solubilisation buffer contains no chaotropes or chaotropes in a concentration of less than 1.5 M urea (c) refolding the solubilised fusion protein and (d) allowing the fusion protein to be cleaved by the N.sup.pro autoprotease moiety under kosmotropic conditions, wherein the recombinant protein of interest is cleaved from the fusion protein, and (e) recovering the protein of interest.

The present disclosure, in some aspects, provides mutant TEV proteases that exhibit improved activity, where the mutant TEV exhibits increase efficiency and/or an increased Kcat for cleavage of an amino acid sequence, and TEV proteases are commonly used for laboratory methods including cleaving fusion proteins and removing a purification tag, such as a maltose binding protein or a poly-histidine tag, from a fusion protein or an antibody.