Choice of fusion proteins, expression host, and analytics solves difficult-to-produce protein challenges in discovery research.

High quality biological reagents are a prerequisite for pharmacological research. Herein a protein production screening approach, including quality assessment methods, for protein-based discovery research is presented. Trends from 2895 expression constructs representing 253 proteins screened in mammalian and bacterial hosts-91% of which are successfully expressed and purified-are discussed.

Reelin is modulated by diet-induced obesity and has direct actions on arcuate proopiomelanocortin neurons.

Objective: Reelin (RELN) is a large glycoprotein involved in synapse maturation and neuronal organization throughout development. Deficits in RELN signaling contribute to multiple psychological disorders, such as autism spectrum disorder, schizophrenia, and bipolar disorder. Nutritional stress alters RELN expression in brain regions associated with these disorders; however, the involvement of RELN in the neural circuits involved in energy metabolism is unknown.

RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes.

Synthetic biology and metabolic engineering seek to re-engineer microbes into "living foundries" for the production of high value chemicals. Through a "design-build-test" cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening.

Elastin-like Polypeptide Diblock Copolymers Self-Assemble into Weak Micelles.

The self-assembly of synthetic diblock copolymers has been extensively studied experimentally and theoretically. In contrast, self-assembly of polypeptide diblock copolymers has so far been mostly studied experimentally. We discovered that the theory developed for synthetic diblock copolymer does not fully explain the self-assembly of elastin-like polypeptide diblock copolymers, leading us to generalize the theory to make it applicable for these polypeptides.

Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest.

Hydration layer coupling and cooperativity in phase behavior of stimulus responsive peptide polymers.

It is shown that hydrophilic (backbone) and hydrophobic (side chain) hydration layers of elastin-like polypeptides (ELPs), a class of stimulus responsive peptide polymers that exhibit lower critical solution temperature (LCST) phase transition behavior, can exist in a coupled and decoupled state. The decoupled hydration state consists of hydrophobic and hydrophilic hydration layers that respond independently to temperature, while the coupled hydration state is characterized by a common, cooperative dehydration of both hydration layers. It is further shown that the primary sequence of an ELP can be tuned to exhibit either of the hydration layer coupling modes.

Calcium binding peptide motifs from calmodulin confer divalent ion selectivity to elastin-like polypeptides.

Calcium-sensitive elastin-like polypeptides (CELPs) were synthesized by periodically interspersing a calcium-binding peptide sequence from calmodulin within an elastin-like polypeptide (ELP) with the goal of creating thermal and calcium responsive peptide polymers. The CELPs exhibit high sensitivity to calcium compared to monovalent cations but do not exhibit the exquisite selectivity for calcium over other divalent cations, such as magnesium, that is displayed by calmodulin. The CELPs were further used as a building block for the synthesis of calcium-sensitive nanoparticles by fusing a hydrophilic, noncalcium-sensitive ELP block with a CELP block that becomes more hydrophobic upon calcium binding.

Predicting transition temperatures of elastin-like polypeptide fusion proteins.

Elastin-like polypeptides (ELPs) are thermally sensitive peptide polymers that undergo thermally triggered phase separation and this behavior is imparted to soluble proteins when they are fused to an ELP. The transition temperature of the ELP fusion protein is observed to be different than that of a free ELP, indicating that the surface properties of the fused protein modulate the thermal behavior of ELPs. Understanding this effect is important for the rational design of applications that exploit the phase transition behavior of ELP fusion proteins.

Self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation.

Chimeric polypeptides (CPs) that are derived from elastin-like polypeptides (ELPs) can self-assemble to form nanoparticles by site-specific covalent attachment of hydrophobic molecules to one end of the biopolymer backbone. Molecules with a distribution coefficient greater than 1.5 impart sufficient amphiphilicity to drive self-assembly into sub-100 nm nanoparticles.

Unexpected multivalent display of proteins by temperature triggered self-assembly of elastin-like polypeptide block copolymers.

We report herein the unexpected temperature triggered self-assembly of proteins fused to thermally responsive elastin-like polypeptides (ELPs) into spherical micelles. A set of six ELP block copolymers (ELP(BC)) differing in hydrophilic and hydrophobic block lengths were genetically fused to two single domain proteins, thioredoxin (Trx) and a fibronectin type III domain (Fn3) that binds the α(v)β(3) integrin. The self-assembly of these protein-ELP(BC) fusions as a function of temperature was investigated by UV spectroscopy, light scattering, and cryo-TEM.

Triple stimulus-responsive polypeptide nanoparticles that enhance intratumoral spatial distribution.

To address the limited tumor penetration of nanoparticle drug delivery vehicles, we report the first pH-responsive polypeptide micelle that dissociates at the low extracellular pH of solid tumors. This histidine-rich elastin-like polypeptide block copolymer self-assembles at 37 °C into spherical micelles that are stabilized by Zn(2+) and are disrupted as the pH drops from 7.4 to 6.

Fusions of elastin-like polypeptides to pharmaceutical proteins.

Elastin-like polypeptides (ELPs) are a class of stimulus-responsive biopolymers whose physicochemical properties and biocompatibility are particularly suitable for in vivo applications, such as drug delivery and tissue engineering. The lower critical solution temperature (LCST) behavior of ELPs allows them to be utilized as soluble macromolecules below their LCST, or as self-assembled nanoscale particles such as micelles, micron-scale coacervates, or viscous gels above their LCST, depending on the ELP architecture. As each ELP sequence is specified at its genetic level, functionalization of an ELP with peptides and proteins is simple to accomplish by the fusion of a gene encoding an ELP with that of the peptide or protein of interest.

Elastin-like polypeptides as a purification tag for recombinant proteins.

This unit presents a recombinant protein purification method that employs an elastin-like polypeptide (ELP) as a purification tag. ELPs undergo a sharp and reversible phase transition when heated above their lower critical solution temperature. ELPs retain this behavior when they are fused to a protein, and thereby provide a simple method to isolate a recombinant ELP fusion protein from cell contaminants by cycling the solution through the insoluble and soluble phase of the ELP fusion protein using a procedure that is termed Inverse Transition Cycling.