Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

• peptide biopolymers that exhibit controlled phase separation based on their amino acid sequence, aromatic:aliphatic ratio, hydrophobicity, temperature, molecular weight, and concentration.
• I DPs Intrinsically disordered proteins
• biological condensates Physical condensates, physically separate themselves from the cytoplasm to control the accessibility of a variety of macromolecules. While our ability to detect protein disorder has advanced rapidly thanks to sophisticated statistical methods, the ability to predict phase separation has lagged behind. The prediction of phase separation is non-trivial, as numerous variables influence phase separation. Broadly, they involve: (1) amino acid composition and amino acid patterning of the primary protein sequence; (2) heterotypic interactions with RNA or other macromolecules; and (3) solvent quality. There are many studies that note the challenge of predicting IDP phase behavior, but few studies that have directly tackled this problem.
• peptide biopolymers comprising intrinsically disordered proteins that exhibit controlled phase separation based on their amino acid sequence, aromatic:aliphatic ratio, hydrophobicity, temperature, molecular weight, and concentration.
• a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising: (X-Z 1 -X-Z 2 -Z 3 - X-Z 4 -Z 3 ) n , where: X is proline (P) or glycine (G) and the ratio of P:G is any number; Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number; Z 2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number; Z 3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number; and Z 4 is tyrosine (Y), histidine (H), tryptophan (W), phenyla
• X is proline (P) or glycine (G) and the ratio of P:G is between 1 :3 and 3:1.
• Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1 :5 and the ratio of K:R can be any number.
• the phase separation is dependent on temperature, molecular weight, hydrophobicity, aromatic:aliphatic ratio, and concentration.
• n is 10 to 200.
• molecular weight is at least 5 kDa to 500 kDa. In another aspect, the molecular weight is about 5 kDa to about 100 kDa.
• the phase separation temperature is 0 to 100 °C. In another aspect, the phase separation temperature is 4 to 25 °C; ⁇ 25 °C; 25 to 37 °C; ⁇ 37 °C; 35 to 38 °C; or >38 °C.
• the polypeptide comprises modified amino acids, a reporter protein, or an enzyme. In another aspect, the sequence comprises: (G-R-G-D- S-P-Y-S)m, where m is 20 to 80.
• the polypeptide comprises a sequence selected from one or more of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 119, 121 , 123, 125,
• compositions comprising a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising: (X-Z 1 -X-Z 2 - Z 3 -X-Z 4 -Z 3 ) n , where: X is proline (P) or glycine (G) and the ratio of P:G is any number; Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number; Z 2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number; Z 3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number; and Z 4 is tyrosine (Y), histidine (H), tryptophan
• X is proline (P) or glycine (G) and the ratio of P:G is between 1 :3 and 3:1.
• Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1 :5 and the ratio of K:R can be any number.
• the composition further comprises an attached molecule comprising one or more of an antibody binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO: 159), an antimicrobial peptide selected from LL37 (SEQ ID NO: 161), lb-M1 (SEQ ID NO: 163), lb-M2 (SEQ ID NO: 165), ib-M5 (SEQ ID NO: 167), Cathelecidin-1 (SEQ ID NO: 169), A(A1 R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA binding proteins (PUM-HD (SEQ ID NO: 159), an
• the composition enhances bioavailability of the attached molecule as compared to the free form of the attached molecule. In another aspect, the composition enhances expression of the attached molecule as compared to the free form of the attached molecule. In another aspect, the composition enhances the stability of the attached molecule as compared to the free form of the attached molecule. In another aspect, the composition enhances stability of the attached molecule during prokaryotic and eukaryotic expression as compared to the free form of the attached molecule. In another aspect, the enhanced stability includes resistance to denatu ration during freezing, thawing, or lyophilization. In another aspect, the composition modulates enzymatic, metabolic, or physiological functions within cells or organisms. In another aspect, the modulation reduces bioavailability of the attached molecules. In another aspect, the attached molecules comprise therapeutic or cytotoxic proteins or peptides.
• Another embodiment described herein is a method for enhancing the bioavailability or stability of a protein, the method comprising creating a fusion protein of one or more proteins and a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising: (X-Z 1 -X-Z 2 - Z 3 -X-Z 4 -Z 3 ) n , where: X is proline (P) or glycine (G) and the ratio of P:G is any number; Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number; Z 2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number; Z 3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T
• X is proline (P) or glycine (G) and the ratio of P:G is between 1 :3 and 3:1.
• Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1 :5 and the ratio of K:R can be any number.
• X is proline (P) or glycine (G) and the ratio of P:G is between 1 :3 and 3:1.
• Z 1 is arginine (R), aspartic acid
• the protein comprises one or more of an antibody binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO: 159), an antimicrobial peptide selected from LL37 (SEQ ID NO: 161), lb-M1 (SEQ ID NO: 163), lb-M2 (SEQ ID NO: 165), lb-M5 (SEQ ID NO: 167), Cathelecidin-1 (SEQ ID NO: 169), A(A1 R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173),
• H5-61-90 SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA binding proteins (PUM-HD (SEQ ID NO: 183), elF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11 D (SEQ ID NO: 189)); KH domains (Yifan or FMRP (SEQ ID NO: 191)); or AAV binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195).
• the enhanced bioavailability of the fusion protein can be used for isolation or separation of a biologic molecule.
• the biologic molecule comprises one or more of a lipid, a cell, a protein, a nucleic acid, a carbohydrate, or a viral particle.
• the nucleic acid is single stranded or double stranded DNA or RNA.
• the viral particle is an adenovirus particle, an adeno-associated virus particle, a lentivirus particle, a retrovirus particle, a poxvirus particle, a measle vims particle, or herpesvirus particle.
• the protein comprises albumin, monoclonal IgG antibodies, or Fc fusion antibodies.
• the isolation or separation is accomplished via reversible phase separation. BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1A-G shows artificial intrinsically disordered polypeptides (A-IDPs) inspired from native idps exhibit reversible UCST phase behavior.
• A-IDPs artificial intrinsically disordered polypeptides
• FIG 1 A shows proteomic analysis of native IDPs that form biomolecular condensates reveal that they have an abundance of G/P, charged and uncharged polar residues, yet exhibit a balance of overall charge.
• FIG 1 B shows an example of a dense, exclusionary phase formed by an UCST exhibiting A-IDP even in the complex medium of bacterial cell lysate.
• FIG. 1 C shows an example SDS-PAGE gel of a set of A-IDPs — [Q5,8]-20 to [Q5,8]-80 — with conserved sequence but increasing MW that show the high purity of the A-IDPs that is obtained by exploiting their UCST phase behavior without need for any chromatographic purification.
• FIG. 1 D shows a visualization of UCST phase separation of [Q5,8]-20 in water-in-oil droplets with fluorescence microscopy.
• FIG. 1 E shows a schematic UCST phase diagram for a cooling-heating cycle of a UCST polypeptide in a water-in-oil droplet.
• FIG. 1 F shows dynamic light scattering data of [Q5,8]-20 demonstrating the change in hydrodynamic radius upon cooling.
• [Q5,8]-20 transitions from soluble unimeric polypeptides with a radius of hydration of 5-6 nm to micron- sized aggregates.
• Data collected at ⁇ 0.0043 in 140 mM PBS, pH 7.4.
• FIG. 2 [WT]-20-sfGFP exhibits phase separation memory upon multiple cycles of heating and cooling. Upon multiple heating and cooling cycles, [WT]-20-sfGFP form puncta in the same location as the first cooling cycle. Given the importance of memory, it is critical to note that the observed transition temperature was below room temperature ( ⁇ 15 °C), suggesting that these cells are naive to phase separation as they were incubated at 37 °C and processed at room temperature. Scale bar indicated 5 ⁇ m. Cooling and heating rate were set to a constant 5 °C min -1 .
• FIG. 3A-B additional proteomic analysis.
• FIG. 3A shows a graph of the difference in amino acid composition between ordered and disordered regions within the same protein.
• FIG. 3B shows a histogram plotting the length of the disordered regions analyzed in this study. Bars indicate 25 th -75 th percentiles and whiskers indicate 10 th -90 th percentiles.
• FIG. 4 SDS-PAGE gels of purified proteins used in this study relevant to FIG. 1-5. Lane labels for each protein purified in this study are listed in Table 2 and Table 3.
• FIG. 6 additional dynamic light scattering data. Data collected on 20 nm filtered samples at volume fractions that were predicted to exhibit liquid-liquid phase separation at 40 °C. Data collected in 140 mM PBS, pH 7.4.
• FIG. 7 cyclic cooling and heating cycles exhibit minimal hysteric behavior.
• Optical turbidity measured at 350 nm of repeated cooling and heating curves of [Q5,8]-20 @ ⁇ 0.0025 between 40 °C and 30 °C.
• FIG. 8A-F show control of UCST cloud point using main chain amino acid composition.
• FIG. 8A shows a schematic describing the methodology for doping repeat unit b into a homopolymer of a.
• GRGDSPYS is doped with increasing fractions of repeat b — GRGDQPYQ — to probe “loss of function" of UCST phase behavior of polymers of a.
• the doping of b into a is designed to ensure mixing of the two repeats along the polypeptide chain and minimize blocky behavior.
• FIG. 8B shows doping of b into a results in mutant IDPs; the UCST cloud point temperature (Tt) of each mutant IDP is a linear function of volume fraction ( ⁇ ) of the A-IDP.
• FIG. 8D shows substitution of aromatic Y residues with aliphatic V dramatically reduces Tt.
• FIG. 8E shows substitution of R with K dramatically reduces Tt.
• a 50% substitution of K for R lowers the Tt by more than 40 °C.
• FIG. 9A-E show effects of single amino acid substitutions on UCST cloud point and new relative UCST propensity scale.
• FIG. 9A-E show effects of single amino acid substitutions on UCST cloud point and new relative UCST propensity scale.
• FIG. 9A shows partial binodal phase boundary of well-mixed, di- block polypeptides with varied ratio of aromatic:aliphatic residues.
• FIG. 9B shows partial binodal phase boundary of well-mixed, di-block polypeptides with varied ratio of polar non-charged residues.
• FIG. 9C shows partial binodal phase boundary of well-mixed, di-block polypeptides with varied identity of positively charged and negatively residues.
• FIG. 9A shows partial binodal phase boundary of well-mixed, di- block polypeptides with varied ratio of aromatic:aliphatic residues.
• FIG. 9B shows partial binodal phase boundary of well-mixed, di
• FIG. 10 shows analysis of secondary structure with circular dichroism (CD) spectroscopy.
• CD spectra of various A-IDPs lack a defined secondary structure curve shape, characteristic of other IDP and other repetitive protein polymers.
• FIG. 11A-C show control of UCST cloud point by molecular weight of A-IDP.
• FIG. 11A shows the molecular weight of the polypeptide affects the Tt.
• FIG. 11 B shows the Tt directly scales with the natural log of MW.
• [WT]-40 has a C sat of ⁇ 1 ⁇ .
• FIG. 12A-D show minor effects on UCST cloud point in protein polypeptides.
• FIG. 12A shows partial binodal phase diagram of sequence syntax permutations focused around the Pro residue. Mutations reveal that amino acid mutation site affects the UCST binodal, particularly at the fifth position, but do not eliminate phase behavior. Data collected under physiologic conditions (140 mM PBS, pH 7.4).
• FIG. 12B shows partial binodal phase boundaries of agnostically non- repetitive but compositionally identical versions of [WT]-20.
• FIG. 12C shows turbidity curves of [H7]-60 in different pH solutions. Decreasing the pH and protonation of the His residues increases and broadens observed UCST phase behavior.
• FIG. 12D shows turbidity curves of [Q5,8]- 40 in solutions with different concentrations of NaCI.
• [Q5,8]-40 exhibits a broad transition at higher temperatures. Increasing the concentration of NaCI between 0-140 mM reduces and sharpens the UCST cloud point, finally reaching a minimum at ⁇ 500 mM. From this point, the protein exhibits a salting-out effect and the transition temperature begins to rise again.
• FIG. 13A-C mapping phase diagrams using a temperature gradient device.
• FIG. 13A shows representative dark-field image of [Q5,8]-20 solutions on a temperature gradient device.
• the transition temperatures of the reference solutions (red and blue lines) and the 20 mg-mL-1 [Q5,8]-20 solution (green line) are indicated by the horizontal colored lines.
• the dashed vertical magenta line along the 20 mg-mL -1 capillary tube illustrated the region of the image used to measure the line scan.
• FIG. 13B shows line scan of normalized light scattering intensity versus temperature for the 20 mg-mL -1 [Q5,8]-20 capillary shown in FIG. 13A.
• the dashed black lines represent tangent lines for the high temperature baseline and increase in light scattering at lower temperatures.
• FIG. 13C shows final binodal phase lines of [WT]-20 and [Q5,8]-20 using multiple data points from temperature gradient device.
• a three-piece fit was utilized to fit three regimes that roughly correspond to the dilute, overlap, and semi-dilute regimes of the polypeptide phase diagram.
• the observed data and subsequent fits demonstrate that polypeptide sequence not only affects UCST cloud point in the dilute regime but over the entire concentration range measured ( ⁇ ⁇ 0.5)
• FIG. 14A-B show quantification of dextran uptake during phase separation of A-IDPs.
• FIG. 14A shows fluorescent microscopy images of phase separated droplets in the presence of dextran molecules of different molecular weight (10/40 kDa) labeled with Alexa488 (green) fluorophore. Inside the phase separated space (dark circles), there is very little sequestration of the dextran molecules as a function of dextran molecular weight or A-IDP sequence. Scale bar is 20 ⁇ m.
• FIG. 14B shows quantification of fluorescent signal between the area inside of phase separated droplets and outside.
• FIG. 15A-G show A-IDPs exhibit tunable intracellular droplet formation based on molecular weight and ratio of aromatic:aliphatic content. All scale bars are 5 ⁇ m.
• FIG. 15A shows a schematic describing the use of two key parameters — ratio of aromatic:aliphatic content and molecular weight — to control intracellular droplet formation by modulating C sat .
• FIG. 15B shows partial in vitro binodal of A-IDP-sfGFP fusions in the dilute regime in 140 mM PBS, pH 7.4. Similar to A-IDPs, A-IDP-GFP fusion proteins exhibit molecular weight and aromatic content dependent phase behavior.
• FIG. 15A shows a schematic describing the use of two key parameters — ratio of aromatic:aliphatic content and molecular weight — to control intracellular droplet formation by modulating C sat .
• FIG. 15B shows partial in vitro binodal of A-IDP-sfGFP fusions in the di
• FIG. 15C shows [WT]-20-sfGFP fusion phase separates in eukaryotic cells (HEK293 cells, Day 5). Instead of forming a single droplet as seen in vitro in protocells (see FIG. 1C), many distinct droplets are formed indicating either diffusion-limited or arrest-limited coalescence.
• FIG. 15D shows confocal fluorescence images of A-IDP-sfGFP as a function of induction time and molecular weight in E. coli. A higher intracellular concentration is required for [WT]-20 versus [WT]-40 to form intracellular droplets. It is noticeable that [WT]-40 has a lower ⁇ ' — A-IDP poor — soluble phase outside the dense droplet phase compared with [WT]-20.
• FIG. 15E shows reducing the aromatic content increases the C sat in a dose-dependent manner.
• FIG. 15F shows A-IDP-sfGFP fusions exhibit a one order of magnitude shift in their C sat as determined by their molecular weight and ratio of aromaticia!iphatic content.
• FIG. 15G shows the size of intracellular droplets ( ⁇ " or dense phase) grow with induction time. As concentration of the A- IDP-sfGFP increases inside the cell, the soluble concentration outside the droplet does not change (FIG. 19) but the size of the intracellular droplets grows relative to the total cell area. Images are individual cells from [3 Y7 : V7]-40-sfG FP cultures at various time points. Error bars represent standard error of the mean.
• FIG. 16A-C show a comparison of partial binodal phase diagrams of A-IDP and A-IDP- sfGFP fusions.
• FIG. 16A shows partial binodal phase boundaries of [WT]-40 and [WT]-40-sfGFP.
• FIG. 16B shows partial binodal phase boundaries of [3Y7:V7]-40 and [3Y7:V7]-40-sfGFP.
• FIG. 15C shows partial binodal phase boundaries of [WT]-20 and [WT]-20-sfGFP. The sfGFP fusion lowers the UCST binodal line for all A-IDPs.
• FIG. 18 shows measurement of total cellular fluorescence as a function of time post induction. E. coli cultures were spun down and resuspended in 140 mM PBS, pH 7.4. The optical turbidity and fluorescence intensity of sfGFP were measured and plotted as a function of time. Data collected at 22 °C.
• FIG. 19 shows measurement of the cellular fluorescence at different locations within the cell. Digital partitions were made between the dense phase separated area of the cell and soluble cytoplasmic space using Imaged. The mean of the total cell fluorescence intensity (solid line) and cytoplasmic fluorescence intensity (dotted line) are plotted as a function of time post-IPTG induction. [WT]-20-sfGFP does not exhibit intracellular droplets until the 6 hr mark. At this point the cytoplasmic fluorescence intensity remains constant but the total fluorescent increases from 6 hr onward. [WT]-40-sfGFP phase transitions prior to the 2-hr timepoint. FIG. 20A-D show A-IDPs exhibit reversible coacervation in E.
• FIG. 20B shows Tt normalized to the intracellular fluorescence of sfGFP in each individual cell
• FIG. 20D shows intracellular binodal lines of various A-IDP-sfGFP fusions. T t increases as a function of cellular fluorescence, a surrogate of A-IDP concentration, and aromatic content of the A-IDP.
• FIG. 20D shows upon reconstitution of sfGFP in the dense phase, the solubility of the reconstituted GFP-A-IDP complex can be modulated with temperature. Data was collected for 36 hr post-IPTG induction and 12 hours post-arabinose induction.
• FIG. 21A-C show image analysis of the number of puncta formed in each cell. 100 cells at random were tabulated for each histogram.
• FIG. 21 A shows a number of intracellular puncta formed in each cell containing [WT]-20-sfGFP during a cooling ramp from 60 °C ⁇ 10 °C (green) and imaged isothermally at 22 °C. Isothermal analysis performed at 6 hours post induction, the first timepoint where intracellular puncta were observed. Cooling ramp performed at 4 hours post induction, where transition temperature (T t ) was between 22 °C and 37 °C.
• T t transition temperature
• FIG. 21 B shows a number of intracellular puncta formed in each cell containing [3Y:V]-40-sfGFP during a cooling ramp from 60 °C ⁇ 10 °C (green) and imaged isothermally at 22 °C. Isothermal analysis performed at 4 hours post induction, the first timepoint where intracellular puncta were observed. Cooling ramp performed at 4 hours post induction, where Tt was between 22 °C and 37 °C.
• FIG. 21 C shows a number of intracellular puncta formed in each cell containing [WT]-40-sfGFP during a cooling ramp from 60 °C ⁇ 10 °C (green) and imaged isothermally at 22 °C. Isothermal analysis performed at 4 hours post induction, the first timepoint where intracellular puncta were observed. Cooling ramp performed at 4 hours post induction, but the transition observed was >37 °C indicating the possibility of memory.
• FIG. 22A-E show engineered intracellular droplets with programmable functions.
• FIG. 22A shows site specific labeling of droplets with a small molecule fluorescent dye.
• DBCO-Alexa488 mixture can diffuse into cells and into the A-IDP condensates within the cell, labeling the azide groups within 10 min of incubation with live £ coli.
• FIG. 22B shows reconstitution of function GFP in condensates by recruitment of a partner from the cytoplasm using a split GFP system.
• a GFP-11-[3Y7:V7]-40 fusion protein is able to recruit GFP-1-10 from the surrounding cytoplasm into intracellular droplets.
• subsequent induction GFP-1-10 by arabinose induction enables recruitment of GFP-10 into the condensates and reconstitution of functional sfGFP within existing intracellular condensates within 12 hr of GFP-1-10 induction (right panel).
• FIG. 22C shows a schematic of enzyme- condensate experiment.
• the a-peptide ( ⁇ p) of LacZ is fused to a fluorescent reporter protein (mRuby3) and expressed from an IPTG-inducible gene from a plasmid in the £. coli strain KRX that has a deletion mutant of the LacZ gene that produces a truncated, catalytically inactive enzyme lacking the op.
• mRuby3 fluorescent reporter protein
• Complementation of Lac ⁇ M15 by a ⁇ p-A-IDP-mRuby3 fusion creates an active enzyme that converts FDG into fluorescein that is then rapidly exported from the intracellular space into the surrounding medium.
• FIG. 22 D shows confocal microscopy images showing the fluorescent conversion of fluorescein Di- ⁇ -D-galactopyranoside (FDG).
• the catalytic efficiency increases with A-IDP MW, as seen by the greater ratio of green fluorescence resulting from FDG conversion to fluorescein normalized to the red fluorescence of mRuby3 on a molar basis.
• Both ⁇ p-[WT]-40-mRuby3 and ⁇ p-[WT]-80-mRuby3 exhibit statistically significant differences from the control ( ⁇ p-mRuby3). Error bars indicate standard error of the mean.
• 22F shows all ⁇ p-A-IDP-mRuby3 fusions exhibit a higher ratio of green fluorescence inside the cell, indicating a greater persistence of fluorescent FDG inside the intracellular space compared to the ⁇ p-mRuby3 control. Error bars indicate standard error of the mean. All scale bars are 5 ⁇ m.
• FIG. 23A-B show confocal microscope images of split GFP recruitment into intracellular droplets.
• FIG. 23A shows GFP-11-[3Y7:V7]-40-mRuby3 co-expressed in the presence of GFP- 1-10 creates fluorescently active GFP only in the interior of the droplet.
• FIG. 24 shows A-IDPs can modulate the solubility of an endogenously bound molecule 2.
• FIG. 25A-B show color balanced confocal microscopy images of ⁇ p-[WT]-40-mRuby3 and ⁇ p-[WT]-80-mRuby3. All scale bars are 5 ⁇ m.
• FIG. 25A shows color re-balanced images from FIG. 22B for improved visualization of the intracellular droplets formed by ⁇ p-A-IDP-mRuby3 fusions.
• FIG. 25B shows split channel images of ⁇ p-[WT]-40-mRuby3 and ⁇ p-[WT]-80-mRuby3.
• FIG. 26 Mender's colocalization score between converted FDG and fluorescent reporter. Data analyzed 30 min after FDG addition. Background threshold was set automatically.
• FIG. 27A-B shows Lineweaver-Burk plots for determining K m and V max .
• Lineweaver-Burk plots created with variable starting concentrations of FDG for FIG. 27A, ⁇ p-mRuby3;
• FIG. 27B ⁇ p-[WT]-20-mRuby3;
• FIG. 27C ⁇ p-[WT]-40-mRuby3;
• FIG. 27D ⁇ p-[WT]-80-mRuby3.
• Slopes (V o ) were determined from fluorescent generation over the course of 20 minutes. Intercepts and slope were used in the calculation of K m and V max .
• FIG. 28A-C show enzymatic droplets formed with variable ratios of aromatic to aliphatic residues. All scale bars are 5 ⁇ m.
• FIG. 28A shows confocal microscopy images observing the fluorescent conversion of Fluorescein Di- ⁇ -D-Galactopyranoside (FDG) of ⁇ p-mRuby3, ⁇ p-[WT]-
• FDG Fluorescein Di- ⁇ -D-Galactopyranoside
• FIG. 28B shows quantified amount of converted FDG intracellularly, normalized to the amount of mRuby3 fluorescence. There is little difference between A-IDPs with different ratios of aromatic:aliphatic content. Error bars indicate standard error of the mean.
• FIG. 29A-B shows enzymatic Activity of ⁇ p-[V7]-40-mRuby3. All scale bars are 5 ⁇ m.
• FIG. 29A shows confocal microscopy images showing the fluorescent conversion of fluorescein Di- ⁇ -D-galactopyranoside (FDG) attached to soluble ⁇ p-[V7]-40-m Ru by3.
• FIG. 29B shows intracellular concentration of fluorescein produced by catalytic conversion of FDG by ⁇ p-[V7]-40- mRuby3, normalized to the mRuby3 fluorescence of each individual cell (n ⁇ 300).
• the soluble fusion exhibits a lower level of enzymatic activity than all puncta-forming ⁇ p-A-IDP fusions, ⁇ p- mRuby3 data from FIG. 24 redrawn to show scale. Error bars indicate standard error of the mean.
• FIG. 30A-D show examples of various fusion proteins that express at low levels in prokaryotic expression systems that when fused to disordered biopolymers rescue expression levels and using the phase separation behavior of the biopolymers allow for recovery into soluble fractions.
• This can be performed with mAb binding proteins that have a nanobody folded structure that bind to mAb (ZD, FIG. 30 A); fluorescent fusion proteins that have beta-barrel structures (sfGFP, FIG. SOB); therapeutic protein peptides (GLP-1 , FIG. 30C) with strong alpha-helical tendencies, RNA binding proteins (PUMHD, FIG. SOD) that have tandem repeat structures and antimicrobial peptides that exhibit cytotoxic tendencies in E. coli.
• FIG. 31 shows the incubation of mAb with a phase separating biopolymer fused to a domain from protein A that binds mAbs.
• the biopolymer is bound to the mAb and centrifuged to capture the mAb heavy (HC) and light chain (LC).
• the supernatant of this step is run in lanes 2 and 5.
• the supernatant is removed, and the pellet is resuspended in an elution buffer that is a lower pH which causes dissociation between the biopolymer-ZD fusion and the mAb.
• the solution is spun again creating an elution supernatant (lanes 3, 6) contains pure mAb HC and LC and other protein contaminants.
• the elution pellet contains the biopolymer and no mAb (lanes 4, 7).
• ZD Z-domain of Protein A
• fusion proteins containing various AMPs were expressed fused to nothing (left side) and various biopolymer (right side).
• fusion protein When fusion protein is not expressed (top), cell growth proceeds as normal measured by an increasing absorbance at ODeoo-
• AMP alone When the AMP alone is expressed (bottom-left), cell growth is stunted (growth curves shifted to later times).
• AMP-biopolymer fusion protein When AMP-biopolymer fusion protein is expressed, normal growth is recovered, suggesting reduced availability of the AMP.
• FIG. 34 shows injection strategies for forming subcutaneous depots in vivo.
• Injection with solubilizing agent such as urea allows for injection under ambient conditions. As the solubilizing agent diffuses faster than the polypeptide, the solvent becomes a poor solvent and the polypeptide will phase separate.
• a dehydrated coacervate can be implanted in the subcutaneous space which will slowly rehydrate indirectly into a two-phase regime.
• FIG. 35 shows fluorescence molecular tomography of (GRGDSPYQ)4o labeled with a near infrared fluorescent dye after injection in the presence of 2 M urea + PBS. Injection concentration of 175 ⁇ , corresponding to 1 .2 mg of protein total.
• FIG. 36 shows fluorescence molecular tomography of (GRGDSPYQ)4o labeled with a near infrared fluorescent dye after injection in dehydrated state. Injection mass equal to 1 .2 mg of protein total.
• each intervening number there between with the same degree of precision is explicitly contemplated.
• the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
• the term “about” as used herein as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
• Binding refers to the binding strength of a binding polypeptide to its target (i.e., binding partner).
• Antagonist refers to an entity that binds to a receptor and activates the receptor to produce a biological response.
• An “antagonist” blocks or inhibits the action or signaling of the agonist.
• An “inverse agonist” causes an action opposite to that of the agonist.
• the activities of agonists, antagonists, and inverse agonists may be determined in vitro, in situ, in vivo, or a combination thereof.
• amino acid refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code.
• Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.
• biomarker refers to a naturally occurring biological molecule present in a subject at varying concentrations that is useful in identifying and/or classifying a disease or a condition.
• the biomarker can include genes, proteins, polynucleotides, nucleic acids, ribonucleic acids, polypeptides, or other biological molecules used as an indicator or marker for disease.
• the biomarker comprises a disease marker.
• the biomarker can be a gene that is upregulated or down regulated in a subject that has a disease.
• the biomarker can be a polypeptide whose level is increased or decreased in a subject that has a disease or risk of developing a disease.
• the biomarker comprises a small molecule.
• the biomarker comprises a polypeptide.
• the terms “control,” “reference level,” and “reference” are used herein interchangeably.
• the reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result.
• Control group refers to a group of control subjects.
• the predetermined level may be a cutoff value from a control group.
• the predetermined level may be an average from a control group. Cutoff values (or predetermined cutoff values) may be determined by Adaptive Index Model (AIM) methodology. Cutoff values (or predetermined cutoff values) may be determined by a receiver operating curve (ROC) analysis from biological samples of the patient group.
• AIM Adaptive Index Model
• ROC analysis is a determination of the ability of a test to discriminate one condition from another, e.g., to determine the performance of each marker in identifying a patient having CRC.
• a description of ROC analysis is provided in P.J. Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of which is hereby incorporated by reference in its entirety.
• cutoff values may be determined by a quartile analysis of biological samples of a patient group.
• a cutoff value may be determined by selecting a value that corresponds to any value in the 25 th -75 th percentile range, preferably a value that corresponds to the 25 th percentile, the 50 th percentile or the 75 th percentile, and more preferably the 75 th percentile.
• Such statistical analyses may be performed using any method known in the art and can be implemented through any number of commercially available software packages (e.g., from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, TX; SAS Institute Inc., Cary, NC).
• the healthy or normal levels or ranges for a target or for a protein activity may be defined in accordance with standard practice.
• expression vector indicates a plasmid, a virus, or another medium, known in the art, into which a nucleic acid sequence for encoding a desired protein can be inserted or introduced.
• host cell is a cell that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector.
• Host cells can be derived from plants, bacteria, yeast, fungi, insects, animals, etc.
• the host cell includes Escherichia coli.
• Polymer as used herein is intended to encompass a homopolymer, heteropolymer, block polymer, co-polymer, ter-polymer, etc., and blends, combinations, and mixtures thereof.
• examples of polymers include, but are not limited to, functionalized polymers, such as a polymer comprising 5-vinyltetrazole monomer units and having a molecular weight distribution less than 2.0.
• the polymer may be or contain one or more of a star block copolymer, a linear polymer, a branched polymer, a hyperbranched polymer, a dendritic polymer, a comb polymer, a graft polymer, a brush polymer, a bottle-brush copolymer and a crosslinked structure, such as a block copolymer comprising a block of 5-vinyltetrazole monomer units.
• Polymers include, without limitation, polyesters, poly(meth)acrylamides, po!y(meth)acrylates, polyethers, polystyrenes, polynorbomenes and monomers that have unsaturated bonds.
• amphiphilic comb polymers are described in U.S.
• amphiphilic comb-type polymers may be present in the form of copolymers, containing a backbone formed of a hydrophobic, water-insoluble polymer and side chains formed of short, hydrophilic non-cell binding polymers.
• polystyrene resin examples include, but are not limited to, polya!kylenes such as polyethylene and polypropylene; polychloroprene; polyvinyl ethers; such as polyvinyl acetate); polyvinyl halides such as polyvinyl chloride); polysiloxanes; polystyrenes; polyurethanes; polyacrylates; such as poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl
• polyacrylamides such as poly(acrylamide), poly(methacrylamide), poly(ethyl acrylamide), poly(ethyl methacrylamide), po!y(N-isopropyl acrylamide), poly(n, iso, and tert-butyl acrylamide); and copolymers and mixtures thereof.
• polymers may include useful derivatives, including polymers having substitutions, additions of chemical groups, for example, alkyl groups, alkylene groups, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art.
• the polymers may include zwitterionic polymers such as, for example, polyphosphorycholine, po lycarboxy beta i n e , and polysulfobetaine.
• the polymers may have side chains of betaine, carboxybetaine, sulfobetaine, oligoethylene glycol (OEG), sarcosine, or polyethyleneglycol (PEG).
• poly(oligoethyleneglycol methacrylate) poly(OEGMA)
• Poly(OEGMA) may be hydrophilic, water-soluble, non-fouling, non-toxic and non-immunogenic due to the OEG side chains.
• Polynucleotide as used herein can be single stranded or double stranded or can contain portions of both double stranded and single stranded sequence.
• the polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine.
• Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.
• a “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds.
• the polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic.
• Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies.
• the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein.
• Primary structure refers to the amino acid sequence of a particular peptide.
• “Secondary structure” refers to locally ordered, three-dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains.
• Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units.
• reporter capable of generating a detectable signal.
• the label can produce a signal that is detectable by visual or instrumental means.
• reporter groups can be used, differing in the physical nature of signal transduction (e.g., fluorescence, electrochemical, nuclear magnetic resonance (NMR), and electron paramagnetic resonance (ERR)) and in the chemical nature of the reporter group.
• Various reporters include signal-producing substances, such as chromagens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like.
• the reporter comprises a radiolabel.
• Reporters may include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein.
• the signal from the reporter is a fluorescent signal.
• the reporter may comprise a fluorophore.
• fluorophores examples include, but are not limited to, acrylodan (6-acryloy 1 -2-dimethylaminonaphthalene), badan (6-bromo-acetyl-2-dimethylamino- naphthalene), rhodamine, naphthalene, danzyl aziridine, 4-[/V-[(2-iodoacetoxy)ethyl]-/V- methylamino]-7-nitrobenz-2-oxa-1 ,3-diazole ester (IANBDE), 4-[/V-[(2-iodoacetoxy)ethyl]-/V- methylamino-7-nitrobenz-2-oxa-1 ,3-diazole (IAN B DA), fluorescein, dipyrrometheneboron difluoride (BODIPY), 4-nitrobenzo[c][1 ,2,5]oxadiazole (NBD), Alexa fluorescent dyes, and derivatives thereof.
• Fluorescein derivatives may include, for example, 5-fluorescein, 6- carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6- tetrachlorofiuorescein, fluorescein, and isothiocyanate.
• Sample or “test sample” as used herein can mean any sample in which the presence and/or level of a target is to be detected or determined. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include a medical sample.
• Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof.
• the sample comprises an aliquot.
• the sample comprises a biological fluid. Samples can be obtained by any means known in the art.
• the sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
• sensitivity refers to the number of true positives divided by the number of true positives plus the number of false negatives, where sensitivity (“sens”) may be within the range of 0 ⁇ sens ⁇ 1.
• method embodiments herein have the number of false negatives equaling zero or close to equaling zero, so that no subject is wrongly identified as not having a disease when they indeed have the disease.
• an assessment often is made of the ability of a prediction algorithm to classify negatives correctly, a complementary measurement to sensitivity.
• specificity refers to the number of true negatives divided by the number of true negatives plus the number of false positives, where specificity (“spec”) may be within the range of 0 ⁇ spec ⁇ 1. Ideally, the methods described herein have the number of false positives equaling zero or close to equaling zero, so that no subject is wrongly identified as having a disease when they do not in fact have disease. Hence, a method that has both sensitivity and specificity equaling one, or 100%, is preferred.
• telomere binding By “specifically binds,” it is generally meant that a polypeptide binds to a target when it binds to that target more readily than it would bind to a random, unrelated target.
• Subject as used herein can mean a mammal that wants or is in need of the herein described peptide biopolymers comprising one or more fusion proteins.
• the subject may be a human or a non-human animal.
• the subject may be a mammal.
• the mammal may be a primate or a non-primate.
• the mammal can be a primate such as a human; a non-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon.
• the subject may be of any age or stage of develo ⁇ ment, such as, for example, an adult, an adolescent, or an infant.
• Transition or “phase transition” refers to the aggregation of the thermally responsive polypeptides. Phase transition occurs sharply and reversibly at a specific temperature called the lower critical solution temperature (LCST) or the inverse transition temperature T A . Below the transition temperature, the thermally responsive polypeptide (or a polypeptide comprising a thermally responsive polypeptide) is highly soluble. Upon heating past the transition temperature, the thermally responsive polypeptides hydrophobically collapse and aggregate, forming a separate, gel-like phase.
• Inverse transition cycling refers to a protein purification method for thermally responsive polypeptides (or a polypeptide comprising a thermally responsive polypeptide). The protein purification method may involve the use of thermally responsive polypeptide's reversible phase transition behavior to cycle the solution through soluble and insoluble phases, thereby removing contaminants.
• Treating” or “treating,” when referring to protection of a subject from a disease, means preventing, suppressing, repressing, ameliorating, or eliminating the disease.
• Preventing the disease involves administering a composition of the present invention to a subject prior to onset of the disease.
• Suppressing the disease involves administering a composition of the present invention to a subject after induction of the disease but before its clinical appearance.
• Repressing or ameliorating the disease involves administering a composition of the present invention to a subject after clinical appearance of the disease.
• “Substantially identical” can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% over a region of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or greater number of amino acids.
• Value refers to the potential binding units or binding sites.
• multivalent refers to multiple potential binding units.
• multimeric and “multivalent” are used interchangeably herein.
• “Variant” used herein with respect to a polynucleotide means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a polynucleotide that is substantially identical to a referenced polynucleotide or the complement thereof; or (iv) a polynucleotide that hybridizes under stringent conditions to the referenced polynucleotide, complement thereof, or a sequences substantially identical thereto.
• a “variant” can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity.
• biological activity include the ability to be bound by a specific antibody or polypeptide or to promote an immune response.
• Variant can mean a substantially identical sequence.
• Variant can mean a functional fragment thereof.
• Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker.
• Variant can also mean a polypeptide with an amino acid sequence that is substantially identical to a referenced polypeptide with an amino acid sequence that retains at least one biological activity.
• a conservative substitution of an amino acid i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids. See Kyte et al., J. Mol. Biol. 1982, 757, 105-132. The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and retain protein function. In one aspect, amino acids having hydropathic indices of ⁇ 2 are substituted.
• hydrophobicity of amino acids can also be used to reveal substitutions that would result in polypeptides retaining biological function.
• a consideration of the hydrophilicity of amino acids in the context of a polypeptide permits calculation of the greatest local average hydrophilicity of that polypeptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity, as discussed in U.S. Patent No. 4,554,101 , which is incorporated herein by reference.
• Substitution of amino acids having similar hydrophilicity values can result in polypeptides retaining biological activity, for example immunogenicity, as is understood in the art.
• Substitutions can be performed with amino acids having hydrophilicity values within ⁇ 2 of each other.
• hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
• a variant can be a polynucleotide sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof.
• the polynucleotide sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof.
• a variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof.
• the amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.
• fusion protein as described herein at least one intrinsically disordered polypeptide and at least one other polypeptide.
• the fusion protein may optionally include at least one linker.
• the intrinsically disordered polypeptide has controlled reversible phase separation.
• the fusion protein includes more than one polypeptide with controlled reversible phase separation.
• the polypeptide with controlled reversible phase separation can include multiple repeats of a peptide motif.
• the fusion protein may include at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, and least 40, at least 60, at least 80, at least 120, at least 160, or at least 200 polypeptides with controlled reversible phase separation or repeats of a peptide motif with controlled reversible phase separation.
• the fusion protein may include less than 30, less than 25, or less than 20 polypeptides with controlled reversible phase separation or repeats of a peptide motif.
• the fusion protein may include between 1 and 160, between 1 and 80, between 1 and 60, between 1 and 40, between 1 and 20, or between 1 and 10 polypeptides with controlled reversible phase separation or repeats of a peptide motif.
• the polypeptides with controlled reversible phase separation may be the same or different from one another.
• the fusion protein includes more than one polypeptide with controlled reversible phase separation positioned in tandem to one another (e.g., repeats of a peptide motif).
• the fusion protein includes one or more binding polypeptide.
• the fusion protein may include at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 binding polypeptides.
• the fusion protein may include less than 30, less than 25, less than 20, less than 10, or less than 5 binding polypeptides.
• the fusion protein may include between 1 and 30, between 1 and 20, or between 1 and 10 binding polypeptides.
• the binding polypeptides may be the same or different from one another.
• the fusion protein includes more than one binding polypeptide positioned in tandem to one another. In some embodiments, the fusion protein includes 2 to 6 binding polypeptides. In some embodiments, the fusion protein includes two binding polypeptides. In some embodiments, the fusion protein includes three binding polypeptides. In some embodiments, the fusion protein includes four binding polypeptides. In some embodiments, the fusion protein includes five binding polypeptides. In some embodiments, the fusion protein includes six binding polypeptides.
• the fusion protein may be expressed recombinantly in a host cell according to one of ordinary skill in the art.
• the fusion protein may be purified by any means known to one of skill in the art.
• the fusion protein may be purified using chromatography, such as liquid chromatography, size exclusion chromatography, or affinity chromatography, or a combination thereof.
• the fusion protein is purified without chromatography.
• the fusion protein is purified using inverse transition cycling. Polypeptides with Controlled Reversible Phase Separation
• the polypeptides with controlled reversible phase separation may comprise any polypeptide that has minimal or no secondary structure as observed by CD, being soluble at a temperature below its lower critical solution temperature (LCST) and/or at a temperature above its upper critical solution temperature (UCST), and comprising a repeated amino acid sequence.
• LCST is the temperature below which the polypeptide is miscible.
• UCST is the temperature above which the polypeptide is miscible.
• the polypeptide with controlled reversible phase separation has only UCST behavior.
• the polypeptide with controlled reversible phase separation has only LCST behavior.
• the polypeptide with controlled reversible phase separation has both UCST and LCST behavior.
• the polypeptide with controlled reversible phase separation may comprise a repeated sequence of amino acids.
• the polypeptides with controlled reversible phase separation may have a LCST between about 0 °C and about 100 °C, between about 10 °C and about 50 °C, or between about 20 °C and about 42 °C.
• the polypeptide with controlled reversible phase separation may have a UCST between about 0 °C and about 100 °C, between about 10 °C and about 50 °C, or between about 20 °C and about 42 °C.
• the polypeptide with controlled reversible phase separation has a transition temperature between room temperature (about 25 °C) and body temperature (about 37 °C).
• a fusion protein comprising one or more thermally responsive polypeptides has a transition temperature between room temperature (about 25 °C) and body temperature (about 37 °C).
• the polypeptide with controlled reversible phase separation has no LCST or UCST behavior.
• the polypeptide with controlled reversible phase separation may have its LCST or UCST below body temperature or above body temperature at the concentration at which the peptide biopolymer comprising one or more fusion proteins is administered to a subject.
• the polypeptide with controlled reversible phase separation comprises one or more thermally responsive polypeptides.
• Thermally responsive polypeptides may include, for example, elastin-like polypeptides (ELP) and resilin-like protein (RLP).
• the polypeptide with controlled reversible phase separation comprises a plurality of polypeptides with controlled reversible phase separation.
• the polypeptide with controlled reversible phase separation a di-block of two or more polypeptides with controlled reversible phase separation.
• the polypeptides with controlled reversible phase separation comprise a di-block of a resilin-like protein (RLP) and an elastin-like polypeptide (ELP).
• the polypeptide with controlled reversible phase separation comprises one or more core polypeptides.
• the core polypeptide is a resilin-like polypeptide (RLP).
• RLPs are derived from arthropod Rec1-resi!in. Red-resilin is environmentally responsive and exhibits a dual phase transition behavior.
• the thermally responsive RLPs can have LCST and UCST. Additional examples of suitable thermally responsive polypeptides are described in U.S. Patent Application Publication Nos. US 2012/0121709, and US 2015/0112022, each of which is incorporated herein by reference.
• the RLP polypeptide comprises the sequence (GRGDSPYS) n (SEQ ID NO: 1).
• the polypeptide with controlled reversible phase separation may comprise an amino acid sequence comprising (G 1 -R2-G 3 -D 4 -S 5 -P 6 -Y 7 -S 8 )) n , where n is 20-200.
• n is 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300.
• n may be less than 500, less than 400, less than 300, less than 200, or less than 100.
• n may be between 1 and 500, between 1 and 400, between 1 and 300, or between 1 and 200. In some embodiments, n is 20, 40, 60, 80, 100, 120, 160, 180, or 200. In one aspect, n is 20 to 200 repeats. In one aspect, n is 20 to 60 repeats.
• Thermally responsive polypeptides may have a phase transition.
• the thermally responsive polypeptide may impart a phase transition characteristic to an unstructured polypeptide or fusion protein.
• Phase transition or “transition” may refer to the aggregation of the thermally responsive polypeptide, which occurs sharply and reversibly at a specific temperature called the lower critical solution temperature (LCST) or the inverse transition temperature (Tt). Below the transition temperature (LCST or Tt), the thermally responsive polypeptides, (or polypeptides comprising a thermally responsive polypeptide) may be highly soluble. Upon heating above the transition temperature, thermally responsive polypeptides hydrophobically may collapse and aggregate, forming a separate, gel-like phase.
• LCST lower critical solution temperature
• Tt inverse transition temperature
• thermally responsive polypeptides can phase transition at a variety of temperatures and concentrations. Thermally responsive polypeptides, for example, may not affect the binding or potency of the binding polypeptides. Thermally responsive polypeptides may allow the fusion protein to be tuned by a user to any number of desired transition temperatures, molecularweights, and formats.
• Thermally responsive polypeptides may exhibit inverse phase transition behavior and thus, the fusion protein comprising the thermally responsive polypeptide may exhibit inverse phase transition behavior.
• Inverse phase transition behavior may be used to form drug depots within a tissue of a subject for controlled (slow) release of the fusion protein.
• Inverse phase transition behavior may also enable purification of the fusion protein using inverse transition cycling, thereby eliminating the need for chromatography.
• X is praline (P) or glycine (G) and the ratio of P:G is any number;
• Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number;
• Z 2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number;
• Z 3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number;
• Z 4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L) and the ratio among Y:H:W:F:M:V:I:A:L can be any number.
• X is proline (P) or glycine (G) and the ratio of P:G is between 1 :3 and 3:1.
• Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1 :5 and the ratio of K:R can be any number.
• the phase separation is dependent on temperature, molecular weight, hydrophobicity, aromatic:aliphatic ratio, and concentration.
• n is 10 to 200.
• molecular weight is at least 5 kDa to 500 kDa. In another aspect, the molecular weight is about 5 kDa to about 100 kDa.
• the phase separation temperature is 0 to 100 °C. In another aspect, the phase separation temperature is 4 to 25 °C; ⁇ 25 °C; 25 to 37 °C; ⁇ 37 °C; 35 to 38 °C; or >38 °C.
• the polypeptide comprises modified amino acids, a reporter protein, or an enzyme. In another aspect, the sequence comprises: (G-R-G-D-S-P-Y-S)m, where m is 20 to 80.
• the polypeptide comprises a sequence selected from one or more of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 119, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143, 145, 147, 149, 151 , 153, 155, 157, or 197-279, or combinations thereof.
• the binding polypeptide may comprise any polypeptide that is capable of binding at least one target.
• the binding polypeptide may bind at least one target.
• “Target” may be an entity capable of being bound by the binding polypeptide.
• Targets may include, for example, another polypeptide, a cell surface receptor, a carbohydrate, an antibody, a small molecule, or a combination thereof.
• the target may be a biomarker.
• the target may be activated through agonism or blocked through antagonism.
• the binding polypeptide may specifically bind the target. By binding target, the binding polypeptide may act as a targeting moiety, an agonist, an antagonist, or a combination thereof.
• the binding polypeptide domain binds
• the binding polypeptide may be a monomer that binds to a target.
• the monomer may bind one or more targets.
• the binding polypeptide may form an oligomer.
• the binding polypeptide may form an oligomer with the same or different binding polypeptides.
• the oligomer may bind to a target.
• the oligomer may bind one or more targets.
• One or more monomers within an oligomer may bind one or more targets.
• the fusion protein is multivalent.
• the fusion protein binds multiple targets.
• the activity of the binding polypeptide alone is the same as the activity of the binding protein when part of a fusion protein.
• the binding polypeptide comprises one or more of an antibody binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO: 159), an antimicrobial peptide selected from LL37 (SEQ ID NO: 161), lb-M1 (SEQ ID NO: 163), lb-M2 (SEQ ID NO: 165), ib-M5 (SEQ ID NO: 167), Cathelecidin-1 (SEQ ID NO: 169), A(A1 R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); ROD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA binding proteins (PUM-HD (SEQ ID NO: 183
• the fusion protein further includes at least one linker. In some embodiments, the fusion protein includes more than one linker. In such embodiments, the linkers may be the same or different from one another.
• the fusion protein may include, none, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or al least 100 linkers.
• the fusion protein may include less than 500, less than 400, less than 300, or less than 200 linkers.
• the fusion protein may include between 1 and 1000, between 10 and 900, between 10 and 800, or between 5 and 500 linkers.
• the linker may be positioned in between a binding polypeptide and a polypeptide with controlled reversible phase separation, in between binding polypeptides, in between polypeptides with controlled reversible phase separation, or a combination thereof.
• Multiple linkers may be positioned adjacent to one another. Multiple linkers may be positioned adjacent to one another and in between the binding polypeptide and the polypeptide with controlled reversible phase separation.
• the linker may be a polypeptide of any amino acid sequence and length.
• the linker may act as a spacer peptide.
• the linker may occur between polypeptide domains.
• the linker may sufficiently separate the binding domains of the binding polypeptide while preserving the activity of the binding domains.
• the linker comprises charged amino acids.
• the linker is flexible.
• the linker comprises at least one glycine and at least one serine.
• the linker comprises at least one proline.
• a vector may include the polynucleotide encoding the fusion proteins detailed herein.
• a vector may include the polynucleotide encoding the fusion proteins detailed herein.
• To obtain expression of a polypeptide one typically subclones the polynucleotide encoding the polypeptide into an expression vector that contains a promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation.
• An example of a vector is pET24. Suitable bacterial promoters are well known in the art.
• a host cell transformed or transfected with an expression vector comprising a polynucleotide encoding a fusion protein as detailed herein.
• Bacterial expression systems for expressing the protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Paiva et al., Gene 1983, 22, 229-235; Mosbach et aI., Nature 1983, 302, 543-545). Kits for such expression systems are commercially available.
• Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are commercially available. Retroviral expression systems can be used in the present invention.
• the fusion protein comprises repeats or single sequences of one or more of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 119, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143, 145, 147, 149, 151 , 153, 155, 157, or 197-279.
• the fusion protein comprises repeats or single sequences of one or more of a polypeptide encoded by a polynucleotide sequence of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, or 158.
• the fusion protein comprises a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, or 316.
• compositions comprising peptide biopolymers comprising one or more fusion proteins can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art to form a therapeutic agent or targeted delivery agent.
• Such compositions comprising peptide biopolymers comprising one or more fusion proteins can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.
• the peptide biopolymers comprising one or more fusion proteins can be administered prophylactically or therapeutically.
• the peptide biopolymer can be administered in an amount sufficient to induce a response.
• the peptide biopolymers are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect.
• An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the peptide biopolymer regimen administered, the manner of administration, the stage, and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.
• the peptide biopolymer can be administered by methods well known in the art as described in Donnelly et al. Ann. Rev. Immunol. 1997, 75, 617-648; Feigner et al., U.S. Patent No. 5,580,859; Feigner, U.S. Patent No. 5,703,055; and Carson et al., U.S. Patent No. 5,679,647, the contents of each of which are incorporated herein by reference in their entirety.
• the peptide biopolymer can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun.
• a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the route of administration.
• the peptide biopolymers can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratu moral, intraperitoneaI, and epidermal routes. In some embodiments, the peptide biopolymer is administered intravenously, intraarterially, or intraperitoneally to the subject.
• the peptide biopolymer can be a liquid preparation such as a suspension, syrup, or elixir.
• the peptide biopolymer can be incorporated into liposomes, microspheres, or other polymer matrices (such as by a method described in Feigner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2 nd ed. 1993), the contents of which are incorporated herein by reference in their entirety).
• Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable, and metabolizable carriers that are relatively simple to make and administer.
• the peptide biopolymer is administered in a controlled release formulation.
• the peptide biopolymer comprises one or more thermally responsive polypeptides, the thermally responsive polypeptide having a transition temperature such that the peptide biopolymer remains soluble prior to administration and such that the peptide biopolymer transitions upon administration to a gel-like depot in the subject.
• the peptide biopolymer comprises one or more fusion proteins comprising one or more thermally responsive polypeptides, the thermally responsive polypeptide having a transition temperature such that the fusion protein remains soluble at room temperature and such that the fusion protein transitions upon administration to a gel-like depot in the subject.
• the fusion protein comprises one or more thermally responsive polypeptides, the thermally responsive polypeptide having a transition temperature between room temperature (about 25 °C) and body temperature (about 37 °C), whereby the fusion protein can be administered to form a depot.
• “depot” refers to a gel-like composition comprising a fusion protein that releases the fusion protein over time.
• the peptide biopolymer can be injected subcutaneously or intratu mo rally to form a depot (coace rvate).
• the depot may provide controlled (slow) release of the peptide biopolymer.
• the depot may provide slow release of the peptide biopolymer into the circulation or the tumor, for example.
• the peptide biopolymer may be released from the depot over a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 1 week, at least about 1 .5 weeks, at least about 2 weeks, at least about 2.5 weeks, at least about 3.5 weeks, at least about 4 weeks, or at least about 1 month.
• the term “detect” or “determine the presence of refers to the qualitative measurement of undetectable, low, normal, or high concentrations of one or more peptide biopolymers, targets, or peptide biopolymers bound to target. Detection may include in vitro, ex vivo, or in vivo detection. Detection may include detecting the presence of one or more peptide biopolymers comprising one or more peptide biopolymers or targets versus the absence of the one or more peptide biopolymer or targets. Detection may also include quantification of the level of one or more peptide biopolymers or targets.
• the terms “quantify,” or “quantification” may be used interchangeably, and may refer to a process of determining the quantity or abundance of a substance (e.g., peptide biopolymer or target), whether relative or absolute. Any suitable method of detection falls within the general scope of the present disclosure.
• the peptide biopolymer comprises a reporter attached thereto for detection.
• the peptide biopolymer is labeled with a reporter.
• detection of a peptide biopolymer bound to a target may be determined by methods including but not limited to, band intensity on a Western blot, flow cytometry, radiolabel imaging, cell binding assays, activity assays, SPR, immunoassay, or by various other methods known in the art.
• any immunoassay may be utilized.
• the immunoassay may be an enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), a competitive inhibition assay, such as forward or reverse competitive inhibition assays, a fluorescence polarization assay, or a competitive binding assay, for example.
• the ELISA may be a sandwich ELISA. Specific immunological binding of the f peptide biopolymer to the target can be detected via direct labels, attached to the peptide biopolymer or via indirect labels, such as alkaline phosphatase or horseradish peroxidase.
• an immobilized peptide biopolymer may be incorporated into the immunoassay.
• the peptide biopolymers may be immobilized onto a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as microtiter wells), pieces of a solid substrate material, and the like.
• An assay strip can be prepared by coating the peptide biopolymer or plurality of peptide biopolymers in an array on a solid support. This strip can then be dipped into the test biological sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
• the present invention is directed to a method of treating a disease in a subject in need thereof.
• the method may comprise administering to the subject an effective amount of the peptide biopolymer comprising one or more peptide biopolymers as described herein.
• the disease may be selected from cancer, metabolic disease, autoimmune disease, cardiovascular disease, and orthopedic disorders.
• the disease is a disease associated with a target of the at least one binding polypeptide.
• Metabolic disease may occur when abnormal chemical reactions in the body alter the normal metabolic process. Metabolic diseases may include, for example, insulin resistance, non- alcoholic fatty liver diseases, type 2 diabetes, insulin resistance diseases, cardiovascular diseases, arteriosclerosis, lipid-related metabolic disorders, hyperglycemia, hyperinsulinemia, hyperlipidemia, and glucose metabolic disorders.
• Autoimmune diseases arise from an abnormal immune response of the body against substances and tissues normally present in the body.
• Autoimmune diseases may include, but are not limited to, lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes melittis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, polyarteritis nodosa, myocarditis, psoriasis, Celiac disease, Crohn's disease, ulcerative colitis, and fibromyalgia.
• Cardiovascular disease is a class of diseases that involve the heart or blood vessels.
• Cardiovascular diseases may include, for example, coronary artery diseases (CAD) such as angina and myocardial infarction (heart attack), stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, and venous thrombosis.
• CAD coronary artery diseases
• Orthopedic disorders or musculoskeletal disorders are injuries or pain in the body's joints, ligaments, muscles, nerves, tendons, and structures that support limbs, neck, and back.
• Orthopedic disorders may include degenerative diseases and inflammatory conditions that cause pain and impair normal activities.
• Orthopedic disorders may include, for example, carpal tunnel syndrome, epicondylitis, and tendinitis.
• Cancers may include, but are not limited to, breast cancer, colorectal cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcomas, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphoid cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, renal cancer, bladder cancer, thyroid cancer, bone cancers, carcinomas, sarcomas, and soft tissue cancers.
• the cancer is colorectal cancer.
• the cancer is colorectal adenocarcinoma.
• the present invention provides a method for using scaffold proteins in developing antibody mimetics for oncological targets of interest.
• scaffold protein engineering come the possibilities for designing potent protein drugs that are unhindered by steric and architectural limitations. Although potent protein drugs can be invaluable for diagnostics or treatments, successful delivery to the target region can pose a great challenge.
• the methods may include administering to the subject a peptide biopolymer comprising one or more fusion proteins as described herein and detecting binding of the peptide biopolymer to a target to determine presence of the target in the subject.
• the presence of the target may indicate the disease in the subject.
• the methods may include contacting a sample from the subject with a peptide biopolymer as described herein, determining the level of a target in the sample, and comparing the level of the target in the sample to a control level of the target, wherein a level of the target different from the control level indicates disease in the subject.
• the disease is selected from cancer, metabolic disease, autoimmune disease, cardiovascular disease, and orthopedic disorders, as detailed above.
• the target comprises a disease marker or biomarker.
• the fusion protein may act as an antibody mimic for binding or detecting a target.
• the methods may include contacting the sample with a peptide biopolymer comprising one or more fusion proteins as described herein under conditions to allow a complex to form between the peptide biopolymer and the target in the sample and detecting the presence of the complex. Presence of the complex may be indicative of the target in the sample.
• the peptide biopolymer is labeled with a reporter for detection.
• the sample is obtained from a subject and the method further includes diagnosing, prognosticating, or assessing the efficacy of a treatment of the subject.
• the method may further include modifying the treatment of the subject as needed to improve efficacy.
• the methods may include contacting a sample from the subject with a peptide biopolymer comprising a fusion protein as detailed herein under conditions to allow a complex to form between the peptide biopolymer and a target in the sample, determining the level of the complex in the sample, wherein the level of the complex is indicative of the level of the target in the sample, and comparing the level of the target in the sample to a control level of the target, wherein if the level of the target is different from the control level, then the treatment is determined to be effective or ineffective in treating the disease.
• Time points may include prior to onset of disease, prior to administration of a therapy, various time points during administration of a therapy, and after a therapy has concluded, or a combination thereof.
• the peptide biopolymer may bind a target, wherein the presence of the target indicates the presence of the disease in the subject at the various time points.
• the target comprises a disease marker or biomarker.
• the peptide biopolymer may act as an antibody mimic for binding and/or detecting a target. Comparison of the binding of the peptide biopolymer to the target at various time points may indicate whether the disease has progressed, whether the diseased has advanced, whether a therapy is working to treat or prevent the disease, or a combination thereof.
• control level corresponds to the level in the subject at a time point before or during the period when the subject has begun treatment, and the sample is taken from the subject at a later time point.
• sample is taken from the subject at a time point during the period when the subject is undergoing treatment, and the control level corresponds to a disease-free level or to the level at a time point before the period when the subject has begun treatment.
• the method further includes modifying the treatment or administering a different treatment to the subject when the treatment is determined to be ineffective in treating the disease.
• compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations.
• the scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described.
• the exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein.
• Clause 1 A polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising:
• X is proline (P) or glycine (G) and the ratio of P:G is any number
• Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number
• Z 2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number
• Z 3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number
• N proline
• G glycine
• K arginine
• K aspartic acid
• K lysine
• K arginine
• R:D is any number and the ratio of K:R can be any number
• Z 2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can
• Z 4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L) and the ratio among Y:H:W:F:M:V:I:A:L can be any number.
• Clause 2 The polypeptide of clause 1 , wherein X is proline (P) or glycine (G) and the ratio of P:G is between 1 :3 and 3:1.
• Clause 3 The polypeptide of clause 1 or 2, wherein Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1 :5 and the ratio of K:R can be any number.
• Clause 4 The polypeptide of any one of clauses 1-3, wherein the phase separation is dependent on temperature, molecular weight, hydrophobicity, aromatic:aliphatic ratio, and concentration.
• Clause 7 The polypeptide of any one of clauses 1-6, wherein the molecular weight is about 5 kDa to about 100 kDa.
• Clause 8 The polypeptide of any one of clauses 1-7, wherein the phase separation temperature is 0 to 100 °C.
• phase separation temperature is 4 to 25 °C; ⁇ 25 °C; 25 to 37 °C; ⁇ 37 °C; 35 to 38 °C; or >38 °C.
• Clause 12 The polypeptide of any one of clauses 1-11 , wherein the polypeptide comprises a sequence selected from one or more of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 119, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143, 145, 147, 149, 151 , 153, 155, 157, or 197-279, or combinations thereof.
• a pharmaceutically acceptable composition comprising a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising:
• X is proline (P) or glycine (G) and the ratio of P:G is any number
• Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number;
• ⁇ 2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number;
• Z 2 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number;
• Z 4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L) and the ratio among Y:H:W:F:M:V:I:A:L can be any number.
• Clause 14 The composition of clause 13, wherein X is proline (P) or glycine (G) and the ratio of P:G is between 1 :3 and 3: 1 .
• Clause 15 The composition of clause 13 or 14, wherein Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1 :5 and the ratio of K:R can be any number.
• Clause 16 The composition of any one of clauses 13-15, further comprising an attached molecule comprising one or more of an antibody binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO: 159), an antimicrobial peptide selected from LL37 (SEQ ID NO: 161), lb-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), lb-M5 (SEQ ID NO: 167), Cathelecidin-1 (SEQ ID NO: 169), A(A1 R, A8R, 117K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA binding proteins (PUM
• Clause 17 The composition of any one of clauses 13-16, wherein the composition enhances bioavailability of the attached molecule as compared to the free form of the attached molecule.
• Clause 18 The composition of any one of clauses 13-17, wherein the composition enhances expression of the attached molecule as compared to the free form of the attached molecule.
• Clause 19 The composition of any one of clauses 13-18, wherein the composition enhances the stability of the attached molecule as compared to the free form of the attached molecule.
• Clause 20 The composition of clause 19, wherein the composition enhances stability of the attached molecule during prokaryotic and eukaryotic expression as compared to the free form of the attached molecule.
• Clause 21 The composition of clause 19 or 20, wherein the enhanced stability includes resistance to denaturation during freezing, thawing, or lyophilization.
• Clause 22 The composition of any one of clauses clause 13-21 , wherein the composition modulates enzymatic, metabolic, or physiological functions within cells or organisms.
• Clause 23 The composition of clause 22, wherein the modulation reduces bioavailability of the attached molecules.
• Clause 24 The composition of clause 23, wherein the attached molecules comprise therapeutic or cytotoxic proteins or peptides.
• a method for enhancing the bioavailability or stability of a protein comprising creating a fusion protein of one or more proteins and a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising:
• X is proline (P) or glycine (G) and the ratio of P:G is any number
• Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number
• Z 2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number
• Z 3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number
• Z 4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L) and the ratio among Y:H:W:F:M:V:I:A:L can be any number
• Clause 27 The method of clause 25 or 26, wherein Z 1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1 :5 and the ratio of K:R can be any number. Clause 28.
• the protein comprises one or more of an antibody binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO:159), an antimicrobial peptide selected from LL37 (SEQ ID NO: 161), lb-M1 (SEQ ID NO: 163), lb-M2 (SEQ ID NO: 165), lb-M5 (SEQ ID NO: 167), Cathelecidin-1 (SEQ ID NO: 169), A(A1 R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA binding proteins (PUM-HD (SEQ ID NO: 159), an antimicrobial peptide selected from
• Clause 30 The method of any one of clauses 25-26, wherein the biologic molecule comprises one or more of a lipid, a cell, a protein, a nucleic acid, a carbohydrate, or a viral particle.
• the nucleic acid is single stranded or double stranded DNA or RNA.
• Clause 32 The method of clause 30, wherein the viral particle is an adenovirus particle, an adeno-associated virus particle, a lentivirus particle, a retrovirus particle, a poxvirus particle, a measle virus particle, or herpesvirus particle.
• Clause 33 The method of clause 30, wherein the protein comprises albumin, monoclonal IgG antibodies, or Fc fusion antibodies.
• phase behavior is encoded in polypeptides.
• Analogous to — and inspired by — synthetic polymers that exhibit lower and upper critical solution temperature (LCST/UCST) phase behavior we began by systematically scanning the sequence space of native IDPs to identify minimal peptide motifs that will confer LCST or UCST phase behavior when polymerized into a macromolecule that consists of many repeats of the peptide motif. With the greatly reduced sequence complexity of these repetitive polypeptides — compared to native IDPs that exhibit LCST/UST phase behavior ⁇ — -we then made rational changes in the amino acid repeat motif that systematically propagate along the sequence.
• repetitive polypeptides can be rationally designed to exhibit both LCST and UCST phase behavior, and their phase behavior can be systematically modulated by amino acid mutations of the repeat motif.
• These artificial polypeptides also exhibit the same basic principles of phase separation inside cells as native IDPs. Informed by a heuristic knowledge of factors that drive phase separation in repetitive polypeptides from these studies as well as the natural composition of membrane-less organelle IDPs, we set out to create artificial IDPs (A-IDPs) that exhibit phase separation in living cells to impart new functionality to the cell.
• A-IDPs from this library to engineer intracellular condensates in living cells.
• the behavior of intracellular condensates for these A-IDPs proved to be surprisingly predictable and tunable, and enabled dynamic control over their cytoplasmic solubility and their interaction with the surrounding environment.
• intracellular droplets capable of sequestering an enzyme whose catalytic efficiency within the engineered condensates can be genetically encoded by modulating the MW of the A-IDP.
• pET24+ vectors were purchased from Novagen (Madison, Wl). gBIock fragments encoding repetitive IDP (A-IDP) sequences of interest, superfolder GFP (sfGFP), mRuby3 and primers for pcDNAS vector were purchased from Integrated DNA Technologies (Coralville, I A). Ligation enzymes, restriction enzymes, DNA ladders were purchased from New England Biolabs (Ipswich, MA). BL21(DE3) chemically competent Escherichia coli (E. coli) cells were purchased from Bioline (Taunton, MA). All E. coli cultures were grown in T errific Broth media purchased from VWR International (Radnor, PA).
• Kanamycin sulfate was purchased from EMD Millipore (Billerica, MA). Protein expression was induced with isopropyl ⁇ -D-1 -thiogalactopyranoside (IPTG) from Gold Biotechnology (St. Louis, MO). All salts, 10/40 kDa fluoresceine labeled dextran molecules, L-(+)-Arabinose, L-Rhamnose and Fluorescein di( ⁇ -D-galactopyranoside) were purchased from Sigma-Aldrich (St. Louis, MO).
• PBS phosphate buffered saline
• EMD Millipore Billerica, MA
• KRX E. coli cell line that endogenously expresses mutated LacZ were purchased from Promega (Madison, Wl).
• NHS Ester reactive fluorophores (NHS-Alexa Fluor® 350 and NHS-Alexa Fluor® 647) were purchased from Life Technologies (Grand Island, NY).
• DNA extraction kits, DNA gel purification kits were purchased from Qiagen Inc. (Germantown, MD).
• Expi293 Eurkaryotic Expression System for HEK293 expression was purchased from Thermo Fischer Scientific (Waltham, MA). Whatman Anotop sterile syringe filters (0.02 ⁇ m) were purchased from GE Healthcare Life Sciences (Pittsburgh, PA). ABIL® EM 90 and TEGOSOFT® DEC surfactants were purchased from Evonik Industries (Essen, Germany). A single emulsion droplet-generating chip was purchased from Dolomite Microfluidics (Royston, United Kingdom). Syringe pumps were acquired from Chemyx Inc. (Stafford, TX).
• Each octapeptide amino acid motif inspired by our proteomic analysis was propagated twenty times in silico. This repetitive amino acid sequence was fed into an algorithm that creates an optimally non-repetitive DNA template from a repetitive protein gene. This 20-mer repeat gene was then ordered from IDT with Gibson assembly overhangs for easy insertion into modified pET24+ vector. To increase the number of total repeats of the gene, we performed iterative cloning steps of Recursive Directional Ligation by Plasmid Reconstruction adding an addition twenty repeats during each step. Transformations were performed into the desired E.
• coli cell line - BL21 (DE3) for recombinant expression and single plasmid confocal experiments and a modified BL21 (DE3) cell line termed KRX by Promega that contains a mutated LacZ gene for enzymatic experimentation.
• genes were inserted into the pBAD33.1 vector by cutting custom pET24+ vector and pBAD33.1 cut with Hind III and Xba I. Gel purification was used to isolate the gene of interest from the housing pET24+ vector, which was then ligated into the similarly cut pBAD33.1 vector. Co-transformation was performed with ⁇ 1 ng final concentration of each plasmid on kanamycin/chloramphenicol dual selection plates.
• Protein was then purified from the insoluble cell suspension fraction.
• cell pellets were isolated by centrifuging cultures at 3500 RCF and resuspending in 20 mL of milli-Q water. Cells were then lysed by sonicating the cell solutions for 2 minutes, with 10 seconds of pulsing followed by 40 seconds of rest on ice (Misonix; Farmingdale, NY).
• the supernatant was collected from this suspension and dialyzed in a 10 kDa membrane (SnakeSkinTM, Thermo Fischer Scientific) against a 1 :200 milli-Q water solution at 4 °C.
• the dialysis water was changed twice over a 48-hour period. From inside the dialysis bag, both insoluble and soluble components were collected and centrifuged at 3500 RCF for 10 minutes and 4 °C. The supernatant was removed and the remaining insoluble pellet containing the protein of interest was lyophilized for a minimum of three days to remove all water from the pellet.
• Protein purity was characterized by 4-20% gradient tris-HCI (Biorad, Hercules, CA) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and staining with either 0.5 M copper chloride or SimplyBlueTM SafeStain (Thermo Fischer Scientific). Protein yield was determined by weight after lyophilization.
• two liquid phases - a dispersed, aqueous phase containing protein of interest in 150 mM PBS and an organic, continuous phase comprised of 75%/5%/20% vol/vol TEGOSOFT® DEC/ABIL® EM 90/mineral oil - were injected into the microfluidic droplet generators at constant flow rates using precision syringe pumps.
• the flow rates of the dispersed and continuous fluids were tuned to ensure droplet formation in the dripping regime; in these experiments, the dripping regime was achieved using a constant flow rate of 500 ⁇ L hr -1 for the organic continuous phase and 50-75 ⁇ L hr -1 for the aqueous, dispersed phase.
• the production of droplets within the microfluidic device was monitored using a 5x objective on an inverted microscope (Leica) equipped with a digital microscopy camera (Lumenera Infinity 3- 1 CCD).
• Circular Dichroism (CD) spectroscopy was performed using an Aviv Model 202 instrument and a 1 mm quartz sample cell (Hellma).
• A-IDPs were prepared by dissolving the purified lyophilized product in 5 mM PBS, pH 7.4 at a final concentration of 10 ⁇ .
• the CD spectra were obtained at 50 °C from 260 nm to 180 nm in 1 nm steps at a 0.5 second average time. Data points with a dynode voltage above 500 V were ignored in the analysis.
• the CD spectra were corrected for the 5 mM PBS buffer signal at 50 °C. This data collection was repeated in triplicate, and the average of the three measurements was represented as molar elIipticity.
• Dynamic light scattering (DLS) measurements were performed over a temperature range of 10-80 °C using a Wyatt DynaPro temperature-controlled microsampler (Wyatt Technology, Santa Barbara, CA). Samples for the DLS system were prepared in 1 x PBS and filtered through 0.02 ⁇ m Whatman Anotop sterile syringe filters (GE Healthcare Life Sciences, Pittsburgh, PA) into a 12 ⁇ L quartz crystal cuvette (Wyatt Technology, Santa Barbara, CA). 5 acquisitions were taken at each temperature for a 5 second duration, and the results presented represent the mean Rh of the sample at each temperature. Temperature-controlled UV-Vis Spectrophotometry
• High concentration A-IDP stock solutions (60 wt%) were prepared by resuspending a mass of lyophilized A-IDP pellets with an appropriate volume of phosphate buffer saline solution (PBS) at a solution pH of 7.0. The concentration was converted to mg mL -1 by assuming that the density of the A-IDP was 1 g mL -1 .
• the RLP stock solution was heated in a water bath at 85 °C for 60 minutes and mixed periodically along with sonication to ensure homogeneity. Lower concentration samples were made by mixing the initial stock solution volumetrically with PBS at a pH of 7.
• TGM temperature gradient microfluidics
• the solutions were loaded into 12 mm x 1 mm x 0.1 mm rectangular borosiIicate glass capillary tubes (VitroCom, Inc.), by capillary action, and sealed with wax to avoid sample evaporation and convection.
• the capillary tubes were held in contact with a hot plate at 85 °C housed within an incubator at 65 °C during the loading process.
• the high temperature environment ensured that the RLP solutions were held above the critical phase transition temperature ( ⁇ 85 °C for [WT]-20).
• Capillary arrays were prepared by taping several capillaries together. The arrays were stored at 85 °C in an oven for 10 minutes prior to subjecting them to the temperature gradient experimentation.
• the temperature gradient device imposed a linear temperature gradient across the A-IDP solutions. This was accomplished by placing the glass capillary array into thermal contact with a heat source on one side and a cold sink on the other. The sample was then bathed in white light. This light was scattered by phase separated A-IDP droplets at cold temperature and was imaged via dark-field microscopy. The temperature gradient was calibrated for each experiment using two reference solutions placed alongside the A-IDP samples of interest.
• the LCST of each reference solution was obtained with a melting point apparatus that measured the light scattering intensity as the temperature was increased at a rate of 0.5 K min -1 .
• the reference solutions became cloudy at temperatures above the LCST.
• LCST was obtained by the onset of light scattering intensity relative to the low intensity baseline on the cold side of the capillary.
• the temperature gradient was calculated using the pixel positions and the LCSTs of the two samples, assuming a linear relationship between position and temperature.
• A-IDPs exhibit roughly a log-normal dependence on UCST cloud point with respect to volume fraction as seen with other repeat polypeptides.
• A coefficients of proportionality
• Cells were grown overnight in 5 mL of TB media from glycerol stocks. In conjunction to fluorescent or confocal imaging, cells were analyzed for total sfGFP fluorescence and OD 600 . Briefly, 50 ul of cell culture at various time points was resuspended in 1 mL of 150 mM PBS. Using a combination of a UV-Vis spectrophotometry signal from a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA) and fluorescent spectra from a NanoDrop 3300 (Thermo Fisher Scientific, Waltham, MA), we calculated the relative ratio of sfGFP fluorescence normalized to cell density.
• a UV-Vis spectrophotometry signal from a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA)
• fluorescent spectra from a NanoDrop 3300
• intracellular pattering of A-IDP-superfolder GFP over time was characterized via fluorescence microscopy using an upright Zeiss Axio Imager D2 microscope with a 20x objective and the appropriate filter set (ex 470/40, em 525/50). Cell fluorescent was calculated using Imaged software. Temperature ramps began at various temperatures but always were set to a constant speed of 5 °C/min.
• [WT]-20-sfGFP was extracted from the pET24(+) vector using polymerase chain reaction (PCR). Briefly, the forward and reverse primers were resuspended with 1 ng of pET24(+) plasmid containing [WT]-20-sfGFP gene fusion. Using a PCR cycle of [98 °C, 1 min; 65 °C, 30 sec; 72 °C, 2 min] x 30 cycles, followed by gel purification, the gene was finally constructed with Gibson assembly.
• pcDNA5 vector containing [WT]-20-sfGFP was transfected into HEK293 cells according to manufacturer instructions (Expi293 Expression System, Thermo Fischer Scientific, Waltham, MA). Cells were spun down at 500 RCF for 10 min at room temperature on day 5 of transient transfection and resuspended in 150 mM PBS for imaging.
• FDG Fluorescein Di- ⁇ -D- Galactopyranoside
• channels were split between fluorescence from FDG and mRuby3 respectively.
• particle analysis tool from Imaged, areas of green fluorescence were isolated from the background. If the mean fluorescence of this area was 5% greater than the background fluorescence (mean fluorescent of the area excluded by the previous particle mask), then this particular particle’s background subtracted green fluorescence was included in the analysis. Particles were excluded if their area was below 0.1 um 2 .
• the background subtracted mean fluorescence of mRuby3 was calculated on the other fluorescent channel. We report the ratio of these two channels as a surrogate for enzymatic efficiency. Error bars are standard errors of the mean at each timepoint.
• FDG Fluorescein Di- ⁇ -D- Galactopyranoside
• channels were first split between fluorescence from FDG and mRuby3 respectively.
• areas of green fluorescence were isolated from the background. If the mean fluorescence of this area was 5% greater than the background fluorescence (mean fluorescent of the area excluded by the previous particle mask), then this particular particle’s background subtracted green fluorescence was included in the analysis. Particles were excluded if their area was below 0.1 um 2 .
• Ratio of fluorescent intensity inside of cells versus the extracellular space is the background corrected mean fluorescence of FDG divided by the background fluorescence. Error bars are standard errors of the mean at each timepoint.
• N, Q, S, T are classified as polar, uncharged amino acids.
• R-K and D- E are pairs of positively charged and negatively charged amino acids.
• G and P are placed into a separate category given their unusual structure and importance in promoting a disordered polypeptide backbone (FIG. 3A).
• the remaining amino acids are classified as “hydrophobic.”
• R and K are both positively charged under normal physiological pH.
• K for R we maintain the charge neutral state of the polymeric backbone — a parameter known to dramatically affect the observed phase behavior.
• N, Q, S and T are all capable of creating hydrogen bonds with water and one another more readily than an aliphatic amino acid such as V.
• substituting these four amino acids for one another maintains an equal number of residues per chain capable for forming this particular type of bond.
• the wild-type (WT) repeat unit is (G 1 -R 2 -G 3 -D 4 -S 5 -P 6 -Y 7 -S 8 ) 40 where 40 refers to the number of repeats.
• the MW of the A-IDPs was varied between ⁇ 15 and ⁇ 70 kDa — by varying the number of repeat motifs from 20 to 80 — to account for observed differences in MW in the intrinsically disordered regions (IDRs) of naturally occurring IDPs (FIG. 3B).
• IDRs intrinsically disordered regions
• the parent sequence is referred to as WT in this paper, and we use a short-hand notation to refer to sequences throughout the text where the bracketed letter refers to a specific point — substitution — mutant.
• a mutant with a complete substitution of Y 7 in the WT repeat unit with V would result in a notation of “[V 7 ]-XX”.
• a residue is only partially substituted in the A-IDP, we use the notation “[BY 0 :ZV 0 ]" where the B to Z ratio represents the ratio of Y to V ratio in the variant and the subscript o is the position of that residue along the repeat unit.
• [Y 7 :V 7 ]-40 would hence represent 50% of all Y replaced with V, whereas [3Y 7 :V 7 ]-40 would represent a 25% substitution of V for Y.
• a double mutant such as 100% substitution of residues at the 5 th and 8 th position in the octapeptide repeat with Q, would be denoted as [Q 5,8 ]-XX with and fractional substitution at these positions with S and Q would be denoted as [BS 5,8 :ZQ 5,8 ]-XX where B and Z represent the ratio of S to Q.
• Full sequence descriptions of common sequences used throughout the paper can be found in Table 1.
• a full description of all architectures of A-IDPs wherein mutant and WT repeats are mixed along the A-IDP chain can be found in Table 2 and Table 3.
• A-IDPs Exhibit Robust and Reversible UCST Phase Behavior in an Aqueous Environment
• One advantage of A-IDPs is their minimal interaction with other proteins or biomolecules stemming from their repetitive nature. This feature of A-IDPs combined with their reversible aqueous two-phase separation enables simple column-free purification by UCST phase transition cycling between the one- and two-phase regime of the phase diagram. An example of this purification process is shown in FIG. 1 B. where the highly expressing A-IDP, [Q 5,8 ]-20. completely phase separates from the soluble fraction of the cell lysate and can be isolated by centrifugation.
• A-IDPs [WT]-20 and [[Q 5,8 ]-20 exhibit UCST phase behavior in vitro.
• These A-IDPs exhibit classic liquid-liquid phase separation where, upon crossing the phase boundary upon cooling from 50 °C to 10 °C, multiple nucleation sites of coace rvate condensates are observed (FIG. 1 D Panel 2).
• Arginine Composition Aromatic to Aliphatic Ratio, Charge Balance and Molecular Weight Define UCST Cloud Point
• A-IDP a set of “mutant” A-IDPs ranging from 100% of a to 100% of b where a is the WT repeat unit.
• the doping scheme wherein the mutant repeat unit b is periodically inserted into the WT sequence is visually illustrated by the color-coded schematic in FIG. 8A.
• the mutant repeat is well mixed — distributed along the WT sequence to reduce “blockiness” of the co-polypeptide, which has been shown in LCST polypeptides to lead to nanoscale self-assembly instead of the desired liquid-liquid coace rvation.
• the T t of the WT and each A-IDP is a linear function of its volume fraction ( ⁇ ) (FIG. 8B and C).
• the linear behavior of the T t of these mutant A- IDPs also allows extrapolation ofthe UCST phase behavior for homopolymers that exhibit a UCST cloud point beyond the experimentally observable range of detection thus putting each point mutation on a single relative scale (FIG. 9).
• phase separation in the presence of low (10kDa) and high (40kDa) MW fluorescently labeled dextran indicate that both [WT]-20 and [Q 5,8 ]-20 droplets are highly exclusionary, as we observed no fluorescence partitioning of dextran into the dense phase (FIG. 14A-B).
• A-IDPs form highly exclusionary droplets in vitro at physiological solution, temperature, and pH conditions ( ⁇ 2 > 0.4).
• A-IDPs have controlled C sat in eukaryotic and prokaryotic cell lines
• each A- IDP was genetically fused to a super folder version of green fluorescent protein (sfGFP) (FIG. 15A).
• sfGFP green fluorescent protein
• coli is significantly different from eukaryotic cells.
• the initiation of the UCST phase transition in E. coli is similar to HEK cells — and in vitro — where small densely fluorescent puncta form after the A-IDP concentration in the cell exceeds C sat, that then grow in size over time (FIG. 15G).
• the growth in the size of these puncta, as more A-IDP is expressed with time, is consistent with measurements of sfGFP fluorescence from the bulk E. coli population normalized to the absorbance at 600 nm ( OD 600 ).
• the increase in fluorescence with time indicates that the intracellular concentration of the A-IDP-sfGFP fusion increases with increased protein induction time (FIG. 18).
• A-IDPs exhibit reversible UCST droplet formation in E. coli Just as one can cross a binodal line into the two-phase regime under isothermal conditions by increasing polypeptide volume fraction, this line may be crossed under constant volume fractions by decreasing solvent quality or the chi parameter ( ⁇ ). Experimentally this is most easily accomplished by reducing the temperature of the bulk solution. Similar to the UCST phase behavior of A-IDPs in vitro, A-IDPs exhibit reversible UCST phase separation inside cells that is reversible by repeated four cooling and heating cycles (FIG. 20A). The phase separation exhibits minimal hysteresis as the difference in the transition temperature of cooling (T t, c) and the transition temperature of heating (TI,H) varies by less than 2 °C (FIG. 20B).
• GFP-11 a Lac operon regulated plasmid that encodes one fragment of GFP (GFP-11) that is fused to [3Y 7 :V 7 ]-40 and a second plasmid regulated by araBAD operon that encodes the other fragment of GFP (GFP-1-10).
• GFP-11- [3Y 7 :V 7 ]-40 at 37 °C proceeds long enough that its intracellular concentrations is greater than itsC sat, we removed the IPTG induction media, and replaced it with arabinose containing media that induce the expression of the larger GFP fragment (GFP-1-10).
• arabinose induction both the ⁇ and ⁇ 2 fractions of the E.
• coli contained fluorescently active GFP (FIG. 22 B). This result suggested that the large GFP fragment is capable of penetrating the preformed condensate in the cell, find its binding partner and form a fully functional molecule, despite the fusion to the A-IDP. Once a fully functional GFP molecule is recruited into the intracellular droplets, it is then possible to dynamically modify the intracellular solubility of the reconstituted GFP-A-IDP by changing the temperature of the bulk (FIG. 24).
• A-IDP fusion that can recruit an enzyme into intracellular droplets to modulate its catalytic activity.
• ⁇ - galactosidase has a range of small molecule substrates, one of which, Fluorescein Di ⁇ -Galactopyranoside (FDG), is colorless but when cleaved by ⁇ -galactosidase, will fluoresce green.
• FDG Fluorescein Di ⁇ -Galactopyranoside
• the DBCO-Alexa488 experiment suggested that a small molecule such as an enzyme substrate can penetrate puncta, even if delivered extracellularly (FIG. 22A).
• the split GFP experiment suggested that relatively large proteins can be recruited to A-IDP condensates to form functional proteins, suggesting that the same should be possible with the split ⁇ -galactosidase system (FIG. 22B).
• This peptide binding system also represents a more ubiquitous, engineered puncta platform as there are a number of split enzyme systems or small protein motifs that have been engineered to bind various intracellular targets.
• FDG Fluorescein Di ⁇ - Galactopyranoside
• A-IDPs that consist of repeats of an octapeptide motif inspired by native IDP exhibit reversible UCST phase separation in aqueous solution. Despite the simplicity of their sequence, they recapitulate many of the features seen in more complex, native I DPs.
• the formation and dynamics of their phase separation into coacervate droplets are controlled by two simple design parameters that are genetically encodable at the sequence level — MW of the A- IDP and the ratio of aromatic:aliphatic residues in the octapeptide repeat. Using these two parameters — aromatic:aliphatic ratio and MW— we were able to produce A-IDPs with C sats ranging from nanomolar to millimolar concentrations.
• A-IDPs phase separate inside cells by the same principles that drive their UCST phase separation in vitro indicating that the same thermodynamic driving forces embedded in the sequence and molecular weight also modulate droplet formation dynamics in isolation. Due to the simplicity of their design, A-IDPs behave in vivo as their phase diagrams in vitro suggest — as their intracellular concentration increases to a C sat , small phase separating droplets form at individual points in space that continue to grow in size with increasing overall A-IDP concentration inside the cell. This predictable observation has been theorized by previous studies but has not been conclusively demonstrated until now.
• these proteins can be used for the de novo design of functional intracellular droplets.
• intracellular puncta capable of binding and recruiting a ⁇ - galactosidase deletion mutant, which could modify the catalytic efficiency of the enzyme-substrate complex — a complex which has not evolved to form intracellular condensates.
• the catalytic efficiency of the reconstituted enzyme in phase separated coace rvate droplets is MW dependent and increases with the MW of the A-IDP. Higher MW A-IDPs more efficiently sequester the substrate in the enzymatically active, intracellular phase separated puncta, which results in a higher catalytic efficiency as measured by K ca t.
• FIG. 30A-D shows examples of various fusion proteins that express at low levels in prokaryotic expression systems that when fused to disordered biopolymers rescue expression levels and using the phase separation behavior of the biopolymers allow for recovery into soluble fractions.
• This can be performed with mAb binding proteins that have a nanobody folded structure that bind to mAb (ZD), fluorescent fusion proteins that have beta-barrel structures (sfGFP), therapeutic protein peptides (GLP-1) with strong alpha-helical tendencies, RNA binding proteins (PUMHD) that have tandem repeat structures and antimicrobial peptides that exhibit cytotoxic tendencies in E. coli.
• ZD nanobody folded structure that bind to mAb
• sfGFP fluorescent fusion proteins that have beta-barrel structures
• GLP-1 therapeutic protein peptides
• PUMHD RNA binding proteins
• FIG. 31 shows the in incubating mAb with a phase separating biopolymer fused to a domain from protein A that binds mAbs.
• the biopolymer is bound to the mAb and centrifuged to capture the mAb heavy (HC) and light chain (LC). Then, the supernatant was removed, and the pellet was resuspended in an elution buffer that is a lower pH which causes dissociation between the biopolymer-ZD fusion and the mAb. The solution was spun again creating an elution supernatant which contains pure mAb HC and LC that had few other protein contaminants. The elution pellet contained the biopolymer and no mAb.
• the first frame shows colocalization of the droplets with a fluorescent signal.
• These biopolymer-fusion proteins retain their liquid-like behavior as droplet fusion occurs between 60- 240 sec.
• FIG. 33 shows fusion proteins containing various AMPs fused to biopolymers.
• peptides The delivery of peptides remains an outstanding challenge for drug delivery.
• protein engineering improvements focused on improving half-life, their effective window lasts from minutes to a few hours rendering them unsuitable for therapeutic use.
• nature utilizes peptides in various biological applications, but regulation of their activity is often tightly controlled by a cellular population that can react to a phenotype change.
• man-made peptide drugs require a delivery solution, one that can improve the pharmacokinetics of these valuable macromolecules.
• sequence engineering such as the incorporation of D-amino acids or other chemically esoteric amino acid derivatives — can limit proteolytic degradation of the protein, but severely limit manufacturing choices. Encapsulation methods produce inconsistent effects on bioavailability or require harsh production conditions that limit the type of peptide drug that can be delivered via these methods.
• GLP-1 is a 31 amino acid peptide produced in the L cells of the intestines, capable of exerting blood glucose control over a large therapeutic window.
• GLP-1 -polypeptide depots revealed much about the design of sub-cutaneous depots for drug release.
• Fusion of a macromolecule such as the polypeptide ELPs reduce potency of the GLP-1 molecule by about ⁇ 30 fold but this does not preclude in vivo activity.
• Zero order release can be achieved for up to 10 days in mice and 17 days in monkeys under optimal conditions.
• Optimal conditions are an injectable transition temperature 5-7 °C below the body temperature of the animal and a molecular weight of 35 kDa or greater to avoid renal clearance.
• GLP-1 The peptide drug of choice for these experiments is GLP-1 for several reasons.
• GLP- 1 can rapidly exert a therapeutic effect in vivo.
• GLP-1 is a prime candidate for improved pharmacokinetics with a half-life of ⁇ 5 min in vivo.
• GLP-1 can be easily studied in established mice models of diet induced obesity where a high fat diet increases the blood glucose.
• GLP-1 is a stable peptide drug which will eliminate confounding variables associated with genetic fusion to various polypeptide partners and myriad delivery strategies.
• transition temperature at injection concentration is the important parameter for determining efficacy.
• both insoluble and soluble components were collected and centrifuged at 3500 RCF for 10 minutes and 4 °C. The supernatant was removed and the remaining insoluble pellet containing the protein of interest was lyophilized for a minimum of three days to remove all water from the pellet.
• Protein purity was characterized by 4-20% gradient tris-HCI (Biorad, Hercules, CA) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and staining with either 0.5 M copper chloride or SimplyBlueTM SafeStain (Thermo Fischer Scientific). Protein yield was determined by weight after lyophilization.
• Temperature dependent UV-vis spectrophotometry Turbidity profiles were obtained for each of the constructs by recording the optical density as a function of temperature (1 °C min -1 ramp) on a temperature-controlled UV-vis spectrophotometer (Cary 300 Bio; Varian Instruments; Palo Alto, CA). The transition temperature (T t ) was defined as the inflection point of the turbidity profile. Samples were measured in PBS at 10 ⁇ M.
• mice were purchased from Jackson Labs (strain 000664) and group housed in a room with a controlled photoperiod (12 hr light/12 hr dark cycle) and allowed at least 1 week to acclimate to the facilities prior to that start of procedures. Animals had unlimited access to water and food and were observed daily for signs and symptoms of distress.
• the diet-induced obese (DIO) phenotype was achieved by maintaining the mice on a high-fat (60 kcal% fat) diet upon arrival to the facility.
• Constructs were endotoxin purified prior to injection by passing the solution through a sterile 0.22 ⁇ m Acrodisc filter comprised of a positively charged and hydrophilic Mustang® E membrane (Pall Corporation). Constructs were filtered in 2 M urea + 140 mM PBS at 37 °C and then dialyzed against milli-Q H 2 O at 4 °C, changing the water three separate times over the course of 72 hours. Aggregated material was removed from the dialysis bag and pelleted with centrifugation (4 °C, 3500 rpm). Samples were frozen and lyophilized for a minimum of 48 hrs.
• the polypeptide is resuspended at 175 ⁇ M in 2 M urea + 140 mM PBS.
• a total volume of 200 ⁇ L is injected into the right hind flank after shaving and removing all hair with chemical dissolution at the site of injection. Mice were weighed to determine injection volume required for ⁇ 2100 nmole of GLP-1 per kg of animal weight. Injection volume did not exceed 200 ⁇ L.
• a small incision is made on the right hind flank with surgical scissors after animals were anesthetized with isoflurane. Incision site was pre-sterilized according to Duke husbandry guidelines. Pre-weighed, dehydrated polypeptide pellets are then inserted under the skin. The pellets rapidly rehydrate and become adherent to the skin tissue and thus for sealing the incision site, only a small amount of surgical glue was used to secure the skin flap. Blood glucose measurement and weight measurements
• mice were put into a clear restraining tube. Their tails were wiped with 50% ethanol in sterile water and then dried. A small incision was made adjacent to the tail vein using a small lancet. The first drop of blood was blotted away. Blood glucose was quantified by applying the second drop of blood to the test strip of an AlphaTRAK 2 blood glucose meter (Abbott Laboratories). Weight was measured on a scale zeroed with a container into which the mice were briefly placed.
• phase behavior of these polypeptide fusions was measured as before with temperature dependent UV-vis spectrophotometry.
• we identify these two proteins indeed have the desired phase behavior with GLP-1-[3Y:V]-20 exhibiting a C sat of ⁇ 30 ⁇ and GLP-1 -[Y:V]-20 exhibiting a C sat of -500 ⁇ (FIG. 37).
• GLP-1- [3V:Y]-20-His6X did not exhibit any phase behavior under physiologic conditions.
• GLP-1 -[3Y:V]-20, GLP-1 -[Y:V]-20 and GLP-1- [3V:Y]-20-His6X were weighed and implanted in the hind flank of C57BI/6J mice that have been fed 60% fat diet.
• the blood glucose data can be visualized in FIG. 38.
• Our strategy of implanting dehydrated depots was successful at controlling blood glucose. It is also positive that we are observing an effect of aromatic:aliphatic ratio, even in non-optimal molecular weight polypeptides.
• Third, our depot forming formulations (GLP-1 -[3Y:Vj-20 and GLP-1-[Y:V]-20) each control blood glucose at least one additional day compared to the soluble RIDP control.
• Body weight measurements also differentiate our two depot forming fusions from one another.
• mice with 2.0 mg depots implanted in their subcutaneous space can be visualized in FIG. 41.
• These proteins that exhibit variable C sat also exhibit variable release from the depot in the subcutaneous space.
• the most hydrophobic depot, GLP-1-S-[40] appears to release the least amount of material suggesting that the depot biophysical properties is retarding a phenotypic effect of the peptide drug.
• GLP-1-[3Y:V]-40 and GLP-1-[Y:V]-4G are still exhibiting weight control at 7 days suggesting that there must be small amounts of material releasing from the depot even at 144 hours after implantation.
• the molecular weight control, GLP-1-[S]-20 exhibits lesser burst released and shorter duration of efficacy than its larger molecular weight analogue, GLP-1-[3Y:V]-40, again supporting the conclusion of delayed entry and prolonged persistence in the blood stream from higher molecular weight depots.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Toxicology (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (2)

Family

ID=77613702

Family Applications (1)

Country Status (5)

Cited By (2)

Families Citing this family (1)

Family Cites Families (7)

• 2021
  • 2021-03-03 AU AU2021231786A patent/AU2021231786A1/en active Pending
  • 2021-03-03 JP JP2022552436A patent/JP2023516653A/ja active Pending
  • 2021-03-03 EP EP21764638.9A patent/EP4087856A4/en active Pending
  • 2021-03-03 WO PCT/US2021/020591 patent/WO2021178483A2/en not_active Ceased
  • 2021-03-03 US US17/908,427 patent/US20230086188A1/en active Pending

Also Published As

Similar Documents

Legal Events