Bioluminescent biosensors useful for monitoring and/or quantifying, in vitro or in vivo, activity of the Hippo signaling pathway. The biosensors monitor LATS kinase activity or YAP-TEAD interaction. The biosensors may be used in methods for monitoring and/or quantifying in real-time, in vitro or in vivo, activity of the Hippo signaling pathway, wherein the activity may be LATS kinase activity and/or YAP-TEAD interaction. The biosensors may be provided in kits for monitoring and/or quantifying in real-time, in vitro or in vivo, activity of the Hippo signaling pathway, wherein the activity may be LATS kinase activity and/or YAP-TEAD interaction.

A bioluminescent single photon bioreactor for performing absolute quantification of light-producing activity by enzymes includes: a bioreactor that produces a bio-electronic signal; an electronic sensor that receives the bio-electronic signal and produces an electrical transduction signal; and an analyzer that receives the electrical transduction signal and absolutely quantifies light-producing activity by enzymes from the electrical transduction signal, such that the absolute quantification is accomplished quantum mechanically by determination of a second order autocorrelation function.

A target protein is prepared as soluble protein using a recombinant protein expression system. An expression vector is used that includes (1) an expression-inducible promoter sequence; (2) a first coding sequence including a polynucleotide coding for a polypeptide that is represented by the formula (Z).sub.n; and (3) a second coding sequence that includes a polynucleotide that codes for a target protein. A method of producing the target protein is also used that includes expressing protein using this expression vector.

The disclosure provides methods for carrying out Real Time Cellular Thermal Shift Assays (RT-CETSA). Also provided are molecular constructs and protein constructs for use in such assays and devices suitable for carrying out such assays.

Disclosed herein are bioluminescent indictors comprising a bioluminescent peptide split by a binding peptide capable of binding a ligand. The bioluminescent indicator bioluminesces upon binding of the binding peptide to its ligand to bring the regions of the bioluminescent peptide into such proximity that the indicator bioluminesces. The bioluminescent indicator may further comprise a leader domain and a transmembrane domain. The bioluminescent indicator acts as a biosensor that can detect and quantify a ligand without the need for an additional light source. Methods for imaging internal body structures are also presented.

The present disclosure is directed to RNA-regulated fusion proteins comprising a protein of interest and an RNA-regulated destabilization domain. Also disclosed are RNA aptamers that bind specifically to a RNA-regulated destabilization domain. Nucleic acid molecules encoding the RNA-regulated fusion proteins and RNA aptamers and methods of use thereof are also disclosed.

The present invention provides a method of designing an optimized gene which comprises altering a nucleotide sequence of a target protein gene, so that only preferential codons with high frequency of use in human cells are selected and a GC content of not less than 60% is achieved. A gene design method which involves the feature “only preferential codons with high frequency of use are selected and a GC content of not less than 60% is achieved” can be established as a general rule for preparing proteins with high expression level, in order to obtain chemically synthesized genes for proteins capable of high-level expression in eukaryotes.

An extracellular vesicle includes an exogenous therapeutic component. The exogenous therapeutic component can include a therapeutic polypeptide, a polynucleotide that encodes a therapeutic polypeptide, a therapeutic nucleic acid, or a therapeutic agent. In some embodiments, the extracellular vesicle includes an exosome or purified exosome product (PEP).

A novel luciferase that is distinct from conventional luciferase has been desired. A luciferase mutant comprising an amino acid sequence in which glutamic acid at position 4, arginine at position 11, leucine at position 18 and valine at position 27 are substituted with other amino acids, in the amino acid sequence of SEQ ID NO: 2.

This disclosure generally relates to synthetic bioluminescent proteins such as synthetic luciferase polypeptides. Methods of making and using the proteins are also provided herein.