Case Studies

In our article on WD40 proteins (Moody, Wolfe, et al., 2021), Steady state flourescence polarization assays were conducted in triplicate 24-point serial dilutions for all six SET1Win peptides against WDR5. Measurements were taken using a SpectraMax i3x plate reader with target peptides tagged with N-terminal sulforhodamine B and C-terminal amides. Steady State FP anisotropy was measured on plates after one hour of incubation in a dark room. Dose response data was graphed and binding affinity for each interaction was determined. This data allowed us to determine the fractional occupancy of binding sites for each of the SET1Win peptides against concentration. Biochem J (2021) 478 (11): 2145-2161

• August 2021

In 2020, we published an article titled “High Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy”, where we outlined novel protocols to uncover the mechanisms of membrane protein and solubilizing detergent interactions. Our protocol uses steady-state fluorescence polarization spectroscopy to observe the rotational mobility of proteomicelle complexes, the formation generated by the binding of membrane proteins with detergent monomers. Labeled membrane proteins and detergents form protein-detergent complexes (PDC) that demonstrate a slowed rotational diffusion with respect to the unbound protein, decreasing the emission in perpendicular planes and increasing emissions in parallel planes to the polarized light. We are able to determine the amount of bound proteins in solution according to this change in emission by analyzing steady state FP anisotropy relative to the equilibrium dissociation constant, and can be used with any membrane protein that forms complexes with detergent micelles. This is an example of an innovational approaches born…

• August 2021

In 2018, we published an article highlighting the creation of a generalizable protein binding scaffold and epitope tag that is orally bioavailable called RPtag. RPtag is based on a ribose-binding protein isolated from an extremophile, awarding it expansive stability across highly variable environmental conditions. It was rationally engineered by splitting an irregular C-terminal pair of beta sheets, generating large and small fragments with high affinity and specificity to each other. We conducted directed mutagenesis to manipulate RPtag specificity by generating 31 mutants of RPtag (large) with a focus on positions proximal to nonconserved residues between RPtag (small) and PDGF-β, a clinically relevant growth factor. Dissocation constant measurements revealed two mutations that improved PDGF-β affinity but not the RPtag (small) sequence; the double mutant variant demonstrated superior affinity, enriching PDGF-β specificity 74x. This is an example of our ability to optimize binding for novel proteins using structure guided rational mutagenesis. Our…

• August 2021

In 2018, we published an article highlighting the creation of a generalizable protein binding scaffold and epitope tag that is orally bioavailable called RPtag. RPtag is based on a ribose-binding protein isolated from an extremophile, awarding it expansive stability across highly variable environmental conditions. It was rationally engineered by splitting an irregular C-terminal pair of beta sheets, generating large and small fragments with high affinity and specificity to each other. Like antibodies, RPtag binds tightly to its epitope, but unlike antibodies, RPtag is small (23kD), allowing it’s use where antibodies fail. In developing RPtag, we established new protocols in fluorescence resonance energy transfer (FRET) assay techniques. Our protocol promotes consistent quantitative analysis of interactions between molecules with nanometer precision. At Ichor Discovery, knowing what questions to ask and acknowledging the limitations of commonly understood techniques endows us a perspective that perpetuates the creation of novel solutions to problems we run…

• August 2021

RPtag is our proprietary orally bioavailable protein binding scaffold. RPtag has two main parts, RPtag (large) and RPtag (small) that have a high binding affinity comparable to that of antibodies. RPtag is designed to be a vehicle for highly specific target delivery and identification, supported by its naturally occurring RBP derivative stability to temperature fluctuations, pH, and proteases. In the creation of RPtag, we developed a surface plasmon resonance (SPR) sensor to capture complexes as an arm of our integrated biochemistry and biophysics pipeline. Our technology allows for direct capture of targets directly from cell lysate and directly perform kinetic characterizations using analytes, subverting excessive handling in laboratory procedures. This method has the potential to enhance target solubility, expression yield, and purity; read more in our publication on RPtag (linked below). At Ichor Discovery, our diligent focus prioritizes the service our clients receive. We are always looking for innovative ways…

• August 2021

Case Study: An Example of Protein Expression and Purification from Yeast In our paper on A2E macular degeneration (Moody, et al., 2016) we synthesized recombinant manganese peroxidase in a P. pastoris strain with an integrated rMnP expression construct isolated from P. chrysoporium. The isolated sequence was cloned into a pGAPZa-A expression vector with an n-terminal alpha factor secretion sequence and a c-terminal myc and histidine tag. The protein was expressed in a 2.8L starter culture and transferred to a 14L Eppendorf bioreactor. The protein was purified in a single step nickel-nitrilotriacetic acid (NiNTA) affinity chromatography column. Production of recombinant manganese peroxidase is a representative example of applied techniques we utilize at Ichor Discovery, allowed us to test rMnP efficacy in breaking down lipofuscin flourophores in the search for treatments of macular degeneration. Read the full article linked below. Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt’s Macular Degeneration…

• August 2021