Synthetic alphavirus-derived replicon expression systems comprising nucleic acid sequences encoding at least one modified nonstructural protein, and synthetic nucleic acid sequences encoding at least one heterologous protein are described. Methods of producing at least one heterologous protein in a cell, or of inducing an immune response in a subject by administering and/or expressing the synthetic alphavirus-derived replicon expression systems are provided.

An insulin degludec derivative and a preparation method therefor are provided. Specifically, a fusion protein has a green fluorescent protein folding unit and an insulin degludec precursor or an active fragment thereof are provided. The fusion protein has a significantly increased expression level, and the insulin degludec precursor protein in the fusion protein is folded correctly, and has biological activity. Moreover, the green fluorescent protein folding unit in the fusion protein can be digested into small fragments by a protease, which have a great difference in molecular weight in comparison to a target protein, and are easy to separate. A method for using the fusion protein to prepare insulin degludec and prepare an intermediate thereof are also provided.

Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence-specificity. A limitation of TEV is its slow catalytic rate, which limits product generation and therefore signal output. Provided is a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is determined via proteolytic release of a membrane-anchored transcription factor, and access to TEV's cleavage site is temporally regulated using a photosensory LOV domain. By gradually decreasing light exposure time, faster variants of TEV were selected over multiple rounds of selection. The mutant TEV proteases and the directed evolution platform are useful in a wide range of biotechnology applications, such as FLARE and SPARK tools.

The technology described herein is directed to regulated synthetic gene expression systems. In one aspect described herein are synthetic transcription factors (synTFs) comprising a DNA binding domain, a transcriptional effector domain, and a regulator protein. In other aspects described herein are gene expression systems comprising said synTFs and methods of treating diseases and disorders using said synTFs.

To program intercellular communication for biomedicine, it is crucial to regulate the secretion and surface display of signaling proteins. If such regulations are at the protein level, there are additional advantages, including compact delivery and direct interactions with endogenous signalling pathways. A modular, generalizable design is provided called Retained Endoplasmic Cleavable Secretion (RELEASE), with engineered proteins retained in the endoplasmic reticulum and displayed/secreted in response to specific proteases. The design allows functional regulation of multiple synthetic and natural proteins by synthetic protease circuits to realize diverse signal processing capabilities, including logic operation and threshold tuning. By linking RELEASE to additional novel sensing and processing circuits, one would be able to achieve elevated protein secretion in response to “undruggable” oncogene KRAS mutants. RELEASE enables the local, programmable delivery of intercellular cues for a broad variety of fields such as neurobiology, cancer immunotherapy and cell transplantation.

Disclosed herein include methods and compositions for making proteins in peroxisomes as well as methods of making cells for producing proteins in peroxisomes. Also disclosed herein are cells for producing a protein in a peroxisome, and methods for producing a protein in a eukaryotic cell containing a peroxisome as described herein.

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.

Described herein are polypeptides, systems, and methods that relate to using domains that bind specifically to a biotinylamide to control receptor and cellular activity.

Aspects of the present disclosure are directed to biosensing circuits that correct crosstalk.

A biosensor comprises first and second molecular components and is capable of displaying non-protease enzyme 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 inhibited non-protease enzyme, whereby the binding event switches the enzyme from a catalytically inactive state to an active state. The second molecular component may comprise a protease that cleaves the first molecular component to release inhibition of the non-protease enzyme of first molecular component. Alternatively, the second molecular component may comprise a trap molecule that binds a bait molecule of the first molecular component to release inhibition of the non-protease enzyme of first molecular component.