WO2024091831A2

WO2024091831A2 - A recombinant spider silk protein and method for tag-free and time-saving purification thereof

Info

Publication number: WO2024091831A2
Application number: PCT/US2023/077242
Authority: WO
Inventors: Hsuan-Chen Wu; Ruei-Ci Wang
Original assignee: National Taiwan University
Priority date: 2022-10-28
Filing date: 2023-10-19
Publication date: 2024-05-02
Also published as: WO2024091831A3

Abstract

The present invention discloses novel recombinant spider silk proteins that may be purified via tag-free and time-saving methods as well as the method for production and purification thereof. More particularly, the present invention discloses amino acid sequences that may be used to produce recombinant spider silk proteins that provide advantages such as self-healing capabilities, tensile properties comparable with native spider silks, high molecular weight, the ability to form micelle-like structures spontaneously, and the ability to be purified using a cost-saving and time-saving method that does not involve tags or diffusion- and/or affinity-based processes, and produces purified proteins which are not denatured and retain a micelle-like form.

Description

A Recombinant Spider Silk Protein and Method for Tag-free and Time-saving Purification Thereof

Field of the Invention

The present invention is in the field of recombinant spider silk proteins, and, more specifically, recombinant spider silk proteins with self-healing property capable of being purified with tag-free, cost-saving and time-saving purification method of the present invention.

Background of the Invention

Spider silk, naturally produced in various forms in the abdominal glands of spiders, is known to be a tough, flexible, and versatile material which further possesses self-healing properties. Among these, the most commonly studied varieties are dragline silks of the golden orb weav er Nephila clavipes and the garden cross spider Araneus diadematus. Such silks are composed mainly of proteins known as major ampullate spidroins (MaSps), which arc comprised of a non-repeating N-terminal domain, a repeating REP domain, and a non-repeating C-terminal domain. Novel recombinant spider silk proteins are designed in the present invention using MaSps of dragline silks of the northern golden orb weaver Nephila pilipes.

As known to those skilled in the art, the extraordinary mechanical properties of spider silk mostly result from the repeat units of polypeptides in the repeating domain forming springlike structures, contributing to the strength, flexibility and other useful properties of silk proteins. As such, recombinant spider silk proteins are commonly designed to exclude the non-repeating N-terminal domain (NTD) and C-terminal domain (CTD). However, we have discovered that spider silk with NT-REP-CT provides advantages such as shortened and simplified purification process as well as self-healing properties.

Specifically, although various recombinant spider silk proteins and methods of production and purification thereof have been previously disclosed, no prior art has disclosed a recombinant spider silk protein that exhibits self-healing properties capable of being purified using a time-saving and cost-saving method comprising based on centrifugation without affinity or diffusion-based processes such as dialysis and chromatography. Most silk proteins known to those skilled in the art are purified using methods which include affinity and/or diffusion-based processes such as dialysis and chromatography, adding not only to production costs but also copious amount of time to the production process. There is, therefore, a need for a purification method for purifying spider silk proteins which excludes such affinity and/or diffusion-based processes and is tag-free, allowing for lower costs, faster purification, and scaling up, and wherein the end product is tag-free, retains a micelle or micelle-like form, and is not denatured.

In addition to its toughness, flexibility, and versatility, certain types of spider silk also possesses self-healing properties. The self-healing properties counteract degradation and wear and tear over time, lending materials created from these spider silks useful in terms of reducing repair and maintenance costs. Due to the difficulty in farming spiders and harvesting natural spider silk, there is also a need for a synthetic spider silk that is easy to produce yet retains the self-healing abilities of native silk.

Summary of the Invention

A recombinant spider silk protein (rSSP) comprising a repeating REP domain wherein the REP domain comprises one or more repeating units wherein each repeating unit comprises R1 protein or R2 protein wherein the amino acid sequence of the R1 protein is at least 80% similar to SEQ ID NO. 3 and wherein the amino acid sequence of the R2 protein is at least 80% similar to SEQ ID No. 4.

A method for purifying the recombinant spider silk protein of claim 2, comprising the steps of: i. lysing a prokaryotic or eukaryotic system comprising the recombinant spider silk protein using a lysis buffer wherein the prokaryotic or eukaryotic system is capable of expressing the recombinant spider silk protein; ii. centrifuging resulting lysis solution to obtain cell pellet; iii. resuspending the cell pellet of step ii in lysis buffer; iv. sonicating the solution of step iii on ice; v. centrifuging the solution of step iv to obtain the cell pellet; vi. resuspending the cell pellet of step v using sodium dodecyl sulfate (SDS) buffer; vii. sonicating the solution of step vi on ice; viii. centrifuging the solution of step vii to obtain the supernatant; ix. centrifuging the supernatant of step viii to obtain the recombinant spider silk protein; and x. washing the recombinant spider silk protein of step ix using deionized (DI) water or ammonium bicarbonate buffer at least 3 times.

Description of the Drawings FIGs. 1 A-1D show transmission electron microscope (TEM) images of the recombinant spider silk proteins NT-MaSpl*32-CT-mEGFP and NT-MaSp2*32-CT-mChcrry expressed in engineered E. coli cells. NT-MaSpl*32-CT-mEGFP is a novel recombinant spider silk protein of the present invention with an NTD comprising the amino acid sequence of SEQ NO. 1, a CTD comprising the amino acid sequence of SEQ NO. 2, a REP domain comprising 32 repetitions of SEQ NO. 3, and an mEGFP protein linked to the C-terminus. NT-MaSp2*32-CT-mCherry is another novel recombinant spider silk protein of the present invention with an NTD comprising the amino acid sequence of SEQ NO. 1, a CTD comprising the amino acid sequence of SEQ NO. 2, a REP domain comprising 32 repetitions of SEQ NO. 4, and a mCherry protein linked to the C-terminus. In FIG. 1A, NT-MaSpl*32-CT-mEGFP is shown in green. In FIG. IB, NT- MaSp2*32-CT-mCherry is shown in red. FIG. 1C is an overlay image of FIG. 1A and IB, demonstrating that both recombinant proteins were produced in the same bacterial cells. FIG. ID is a bright field image of the individual E. coli cells. It is apparent from these four images that the recombinant proteins NT-MaSpl*32-CT-mEGFP and NT-MaSp2*32-CT-mCherry were expressed simultaneously in the same individual E. coli cells.

FIGs. 2A-2D show transmission electron microscope (TEM) images of the recombinant spider silk proteins NT-MaSpl*32-CT-mEGFP and NT-MaSp2*32-CT-mCherry purified from engineered E. coli cells. In FIG. 2A, NT-MaSpl*32-CT-mEGFP is shown in green. In FIG. 2B, NT-MaSp2*32-CT-mCherry is shown in red. FIG. 2C is an overlay image of FIG. 2A and 2B, demonstrating the presence of both recombinant proteins in the same micelle-like structures. FIG. ID is a bright field image of the purified recombinant proteins. It is apparent from these four images that the recombinant proteins NT-MaSpl*32-CT-mEGFP and NT-MaSp2*32-CT- mCherry were co-localized in micelle-like structures and remained so after purification.

FIG. 3A shows a sample of NT-MaSpl*32-CT stretched and molded for mechanical testing. The thickness of the silk sample is about 10.67 pm. FIG. 3B is a graph demonstrating the toughness of NT-MaSpl *32-CT, with true strain in percentages as the x-axis and true stress (also known as true strength) in MPa as the y-axis. The definitions of stress and strain arc as commonly known to those skilled in the art. The red, green, and blue lines represent three replicated tests. Each spun silk fiber, pre-mounted on a paper holder, was clamped onto a tensile tester via the sample grips. Subsequently, the mechanical performance of each fiber was measured and recorded to generate the final tensile curves. The tensile toughness of NT- MaSpl*32-CT, calculated from the area under the average strcss-strain curve, is 38.63 ± 15.93 MJ/m³.

FIG. 4A shows a sample of NT-MaSp2*32-CT stretched and molded for mechanical testing. The thickness of the silk sample is about 13.83 pm. FIG. 4B is a graph demonstrating the toughness of NT-MaSp2*32-CT, with true strain in percentages as the x-axis and true strength in MPa as the y-axis. The red, green, and blue lines represent three replicated tests. Each spun silk fiber, pre-mounted on a paper holder, was clamped onto a tensile tester via the sample grips. Subsequently, the mechanical performance of each fiber was measured and recorded to generate the final tensile curves. The tensile toughness of NT-MaSp2*32-CT, calculated from the area under the average stress-strain curve, is 63.53 ± 10.91 MJ/m³.

FIG. 5 A shows a sample of NT-MaSp2*96-CT stretched and molded for mechanical testing. The thickness of the silk sample is 14.05 pm. FIG. 5B is a graph demonstrating the toughness of NT-MaSp2*96-CT, with true strain in percentages as the x-axis and true strength in MPa as the y-axis. The red, green, and blue lines represent three replicated tests. Each spun silk fiber, pre-mounted on a paper holder, was clamped onto a tensile tester via the sample grips. Subsequently, the mechanical performance of each fiber was measured and recorded to generate the final tensile curves. The tensile toughness of NT-MaSp2*96-CT, calculated from the area under the average stress-strain curve, is 180.35 ± 7.45 MJ/m³, similar to that of native spider silks.

FIGs. 6A and 6B show negatively stained TEM images of the recombinant spider silk protein NT-MaSpl*32-CT in the form of micelles each in different magnifications as indicated by the scale of each photograph. The approximate micelle size of NT-MaSpl*32-CT is about 150 to 600 nm.

FIGs. 7A and 7B show negatively stained TEM images of the recombinant spider silk protein NT-MaSp2*32-CT in the form of micelles each in different magnifications as indicated by the scale of each photograph. The approximate micelle size of NT-MaSp2*32-CT is about 150 to 600 nm. FIGs. 8 A and 8B show negatively stained TEM images of the recombinant spider silk protein NT-MaSp2*96-CT in the form of micelles each in different magnifications as indicated by the scale of each photograph. The approximate micelle size of NT-MaSp2*96-CT is about 150 to 1,000 nm.

FIG. 9 shows the ratio of 339 nm/351 nm fluorescence emission, used to monitor changes in protein structure, of NT-MaSpl*32-CT (a recombinant spider silk protein with an NTD and CTD of the present invention, and 32 repeats of SEQ NO. 3 as the REP domain), and NTCR- MaSpl*32-CT (a recombinant spider silk protein with NTD modified at D35K and K60D, a CTD of the present invention, and 32 repeats of SEQ NO. 3 as the REP domain) at different pH levels in the form of a line graph, demonstrating pH dependent silk NTD-dimerization using tryptophan fluorescence signal change as indicator. As shown in FIG. 9, fluorescence ratio of recombinant spider silk proteins with unaltered NT domains decreased at pH levels lower than about 7.6 to 7.2, while fluorescence ratio of recombinant spider silk proteins with mutated NTCR domains remained stable at pH levels of 6 to 8.8, indicating structural stability at different pH levels.

FIG. 10A to 10C show fluorescence microscopy imaging of results of cell-free protein synthesis (CFPS). FIG. 10A shows fluorescence microscopy imaging of the positive control group, CFPS used only to produce mEGFP. As demonstrated in the figure, the green fluorescence of mEGFP was evenly distributed. FIG. 10B shows fluorescence microscopy imaging of NT-MaSpl*32-CT-mEGFP. As demonstrated in the figure, green fluorescence, representing the recombinant protein of the present invention with mEGFP attached for ease of observation, was disordered and aggregated. FIG. 10C shows fluorescence microscopy imaging of NTcR-MaSpl*32-CT-mEGFP. As demonstrated in the figure, green fluorescence was present in a particulate, micelle-like form.

FIG. 11 shows the result of dynamic light scattering (DLS) analysis of NTcR-MaSpl*32- CT-mEGFP produced using CFPS with diameter in nm as the x-axis and frequency in percentages as the y-axis. The resulting Z-average was 146.5 nm, while the PI was 0.462.

FIGs. 12A-12D show fluorescence microscopy imaging of the recombinant spider silk proteins of the present invention, with NTcR-MaSpl*32-CT-mEGFP in green in FIG. 12A, NTcR-MaSp2*32-CT-mCherry in red in FIG. 12B, FIG. 12A and 12B overlaid in FIG. 12C, and the bright field image in FIG. 12D. As demonstrated in the figures, the two recombinant spider silk proteins produced using CFPS still spontaneously assemble into the same miccllc-likc structures.

FIG. 13 shows the results of tensile testing with strain in percentages as the x-axis and stress in N/mm² as the y-axis. The black line represents the strcss-strain curve of uncut NT- MaSp2*96-CT film and treated only with ethanol while the red line represents NT-MaSp2*96- CT cut, soaked in ethanol and had 1.3 MPa pressure exerted upon it. As demonstrated in the figure, healing of the recombinant spider silk film occurs with the aid of water and pressure, and can recover some mechanical properties. Notably, the location of fracture on the cut and healed film sample was different from the location of adhesion, which shows that the recombinant spider silk protein of the present invention exhibits self-healing properties.

FIGs. 14A and 14B show self-healing of two fibers produced using the novel recombinant spider silk protein of the present invention, CoRl/R2, were wound together using a black string with hair clips used to hold both ends of the string. The fibers before healing are shown in FIG. 14A. FIG. 14B shows the results of hot water treatment. As demonstrated in the photo, the two fibers have healed up into one.

FIG. 15 illustrates an exemplary method of purifying the recombinant spider silk protein of the present invention.

FIGs. 16A and 16B show SDS-PAGE analyses of purified recombinant spider silk proteins have purity of up to or above 98%.

FIG. 17A shows change of incision width after healing process for both Nephila pilipes and Cyrtophora moluccensis major-ampullate dragline silk, indicating intrinsic material repairability of native spider silks. FIG. 17B shows the change of crack thickness as an index of spider silk film healing capacity. The width difference of spider silk films was normalized to its original gap thickness and plotted. Both FIG. 17A and 17B show that film produced using N. pilipes silk demonstrated superior healability to that of C. moluccensis.

FIG. 18 illustrates graphene spider silk films capable of conducting electricity. Films containing recombinant spider silk protein of the present invention as well as 5%, 10%, and 15% graphene were produced as a potential bioelectronic scaffold. Film samples were cut and allowed to heal, with conductivity tests done before cutting and after healing. Uncut graphene spider silk films could conduct electricity, lighting up LED lights. Healed graphene spider silk films could also conduct electricity, lighting up LED lights.

FIG. 19 further demonstrates the healability of spider silk films produced using the recombinant spider silk protein NT-MaSp2*32-CT. The cut and healed spider silk films exhibited comparable mechanical strength to that of the uncut films.

FIGs. 20A and 20B illustrate healing capability of NT-MaSp2*32-CT silk films (natively cast in HFIP). The FIG. 20 A histogram shows the stress that can be endured by silk films posttreatment and after healing, with original silk film, film treated with 100% EtOH, film treated with 70% EtOH, film treated with 37°C water vapor annealing for 2 hrs, film treated with 60°C water vapor annealing for 1 hr, as well as film treated with 60°C water vapor annealing for 2 hrs, followed by film-healing challenges. Black bars show the effect of post-treatments on mechanical strength of the films, while gray bars show the corresponding healing capability. Both 37°C (2 hrs) and 60°C (1 hr) treatments rendered exceptional mechanical performance after healing. FIG. 20B graphs self-healed stress and strain of cast silk materials and film produced using the recombinant spider silk protein NT-MaSp2*32-CT. Films treated under 60°C water vapor annealing (1 hr) exhibited multiple-healing/reversible-healing capacities. The same treated films were repetitively self-healed and separated 1 to 5 times, and results show no significant changes in mechanical performance (mechanical performance after the 5^th healing was slightly reduced but still demonstrated good mechanical performance). Neither strength (shown in the histogram in blue) nor elasticity (shown in the histogram in red) were affected.

FIGs. 21A and 21B show fabrication and applications of treated silk materials produced from the recombinant spider silk protein NT-MaSp2*32-CT. Films treated with 37°C water vapor annealing were woven and healed in various dimensional configurations, as shown in FIG. 21 A. Both silk film strips (the black being graphene doped silk samples, the transparent being pure silk samples) could form intertwined structures in ID, 2D, and 3D after the healing process. FIG. 2 IB shows that fabricated silk strips (treated with 60°C water vapor annealing for 1 hr) were able to adhere, forming configurable rings with adjustable sizes. The rings were reopened and self-healed repetitively. The same silk-based ring was readjusted to fit either the thumb or the little finger, conferring a wearable device as a smart resistance sensor for sensing human fingers or skin. The device is set as a finger/skin recognition system using electrical resistance, turning “on” only when fingers arc detected, and remaining “off’ when the resistance is too low or too high.

FIGs. 22A and 22B show repairability/healability of silk films produced using native spider silk and mulberry silk worm silk. The silks of orb-weaver spider Nephila pilipes, tentbuilder spider Cyrtophora moluccensis, and mulberry silkworm Bombyx mori were used, with major-ampullate dragline silks collected from the two spiders and silk samples harvested from degummed cocoons of B. mori. The precast silk films from both spiders (2.5% w/v in HFIP) were cut by a razor blade to introduce rifts. The incision width of the cracked region on both silk films was measured before and after addition of water droplets. FIG. 22A shows change of incision width after the healing process, demonstrating the intrinsic material repairability of silk materials harvested from the spiders and the silkworm. FIG. 22B shows the change of crack width as an index of silk film healing ability. The width difference of silk films was normalized to its original gap thickness and plotted using the formula shown in the figure and plotted. The silk film of N. pilipes showed superior healability to those of B. mori and C. moluccensis.

FIG. 23 shows the ratio of R1:R2 recombinant spider silk proteins in films of Example 15. R1 is represented in pink, while R2 is represented in blue. R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.

FIG. 24 illustrates results of water resistance test on films created by mixing different ratios of R1 and R2 recombinant spider silk proteins. Test results show that 100:0 pure R1 films retain about 85.1% of their original mass. 0:100 pure R2 films completely dissolved in water. Films with higher proportions of R1 exhibit higher levels of water resistance. 75:25 films retained 83.3% of their original mass; 50:50 films retained 81.7% of their original mass; 33:67 films retained 81.2% of their original mass; 25:75 films retained 80.7% of their original mass, and 10:90 films retained 50.3% of their original mass. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 25 illustrates results of FTIR analysis on recombinant spider silk films with R1:R2 of about 0: 1, about 1:3, about 1: 1, about 3: 1, and about 1:0. The yellow dotted line marks 1,622 cm ¹; a peak at this wavenumber represents the presence of -sheets in the material. The blue dotted line marks 1,649 cm ¹; a peak at this wavenumber represents the presence of amorphous structures beta-turn, beta-spiral and 3i-helix in the material. The proportion of the number of 3 - sheets and amorphous structures in each film can be approximated by calculating the ratio of these two peaks. Results indicate that R1 spider silk indeed comprises more 3 -sheet structures than does R2, and that the proportion of /3 -sheet structures within R1 and R2 mixture films is positively correlated with the R1:R2 ratio. As such, films with higher proportion of R1 should exhibit higher water resistance and tensile strength, properties related to the presence of /? -sheet structures. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 26 illustrates water absorption of recombinant spider silk films with R1:R2 ratio of 1:0, 3: 1, 1: 1, 1:2, and 1:3. Water absorption is represented by percent weight gain. As shown in the graph, 1:0 pure R1 film exhibits lowest water absorption, while 1:3 film exhibits highest water absorption. Water absorption could not be measured for pure R2 film, which dissolved in water. 1:0 film absorbed 82.0% its mass, 3: 1 film absorbed 99.6%, 1: 1 film absorbed 112.3%, 1:2 film absorbed 172.7%, and 1:3 film absorbed 337.0%. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 27 illustrates results of tensile testing for pure R1 and pure R2 films. FIG. 27 A illustrates results of tensile testing on R1 film, with strain (%) on the x-axis and strength (N/mm²) on the y-axis. FIG. 27B illustrates results of tensile testing on R2 film. FIG. 27C compares the strength of R1 film and R2 film. As shown in the graphs of FIG. 27, the average maximum stress of R1 film is 38.5 N/mm², while the maximum stress of R2 film is 30.8 N/mm². It can be observed that R1 film exhibits higher tensile strength than R2. In terms of ductility, R1 film does not exceed 5%, and the ductility of R2 film is averaged at 10%. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 28 illustrates the self-healability testing method of Example 19. The original film group is cut into appropriate shape and size, then subjected to tensile tests to analyze tensile strength. The healed film group (also known as self-healing film group) is cut into appropriate shape and size, slashed, sclf-hcalcd, and dried prior to tensile testing. In other words, films of the healed film group are cut in half, the area to be healed is dipped in deionized water, and the dipped portions of the cut films are overlapped for self-healing. Films of the healed film group are placed in a dry cabinet. After tensile testing, the healed films are inspected to ensure that the new rips did not occur in the self-healed area. Only healed films with stock-break failure separations following tensile testing are considered to have been effectively healed. Tensile strength is calculated. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 29 illustrates healability of recombinant spider silk films with R1:R2 of about 1:0, about 3: 1, about 1:1, about 1:2, about 1:3, and about 0: 1. FIG. 29A illustrates the results of tensile testing for both original films and healed films of the various R1:R2 ratios. In the original film group, tensile strength of pure R1 film is measured at 38.5 N/mm², 3: 1 film at 35.5 N/mm², 1: 1 film at 33.8 N/mm², 1:2 film at 32.7 N/mm², 1:3 film at 32.1 N/mm², and pure R2 film at 30.8 N/mm². In the healed film group, tensile strength of pure R1 film is measured at 14.4 N/mm², 3: 1 film at 24.2 N/mm², 1:1 film at 28.4 N/mm², 1:2 film at 30.9 N/mm², 1:3 film at 31.4 N/mm², and pure R2 film at 30.3 N/mm². FIG. 29B illustrates healability of recombinant spider silk films of different R1:R2 ratios. After tensile testing of healed films, all but the films with stock-break failure are eliminated, and healability is calculated by dividing strength of healed film by strength of original film. As shown in FIG. 29B, pure R1 film exhibits lowest healability, recovering only 37.4% tensile strength, while pure R2 film exhibits highest healability at 98.3% tensile strength. The healability of 3:1 film is 68.2%, 1:1 film is 84.1%, 1:2 film is 94.5%, and 1 :3 film is 97.7%. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 30 illustrates the method for creating a one-layer protein film pipe used in Example 20. Using recombinant spider silk film of R1 :R2 = 1 :3, which exhibits highest healability and is insoluble in water, a protein film pipe is created by rolling the film and allowing it to self-heal. Tests are performed to examine the protein film pipe’s application as a water pipe. A hole is cut into the pipe to simulate damage in the protein film pipe, and a new piece of film is used to patch the damaged section. After patching, water is allowed to flow through the pipe in order to examine the protein film pipe’s water resistance as well as results of patching. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 31 illustrates the protein film pipe of Example 20 created from one layer of recombinant spider silk film. Thickness of the film is 200 fl m, the inner diameter of the pipe is 4.5 mm, and the flow of water is set to 150 mL/min. As shown in FIG. 31 A, spider silk film is rolled into a protein film pipe, and some water is applied to the overlapping region to facilitate self-healing. Afterwards, the pipe is allowed to dry in a dry cabinet for at least 1 day. Water flow devices are created as shown in FIG. 3 IB, wherein the protein film pipe is placed in the center, and metal pipes are used to connect the protein film pipe to plastic pipes. A peristaltic pump is used to continuously pump water into the pipes, with water flow set to 150 mL/min. The pipes are observed for 1 hr. Within 30 min, the protein film pipe is observed to contract inwards, although no leakage or damage to the pipe has been observed, and the protein film pipe does not contract further. This contraction is attributed to the film becoming softer after absorbing water, as well as structural instability. FIG. 31C demonstrates the flow of water within the pipe system. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 32 illustrates the method for creating a two-layer protein film pipe used in Example 21. Using recombinant spider silk film of R1:R2 = 1:3. one layer of film is rolled into a cylindrical shape, then a second layer of film is used to cover the first, with some water applied to facilitate healing into pipe-shape as well as healing between the first and second layers. The two-layer protein film pipe is placed in a dry cabinet for at least 1 day. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 33 illustrates the protein film pipe (two-layer) of Example 21. As shown in FIG. 33A, the total thickness of the film is 400 /z m, the inner diameter of the pipe is 4.5 mm, and the flow of water is set to 150 mL/min. The pipes are observed for 2 hrs, during which no contraction or obvious shape change is observed, as illustrated in FIG. 33B. FIG. 33C illustrates the flow of water through the protein film pipe. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

FIG. 34 illustrates the process of testing the healing capabilities of the two-layer protein film pipe of Example 21. As shown in FIG. 34 A, a hole is cut into the two-layer protein film pipe to simulate possible damage to the pipe. The diameter of the puncture is about 1 mm. When water flow is turned on, leakage is observed from the pipe. Then a piece of new film 100 fl m thick, 3 mm by 3 mm in area is used to patch the hole, as shown in FIG. 34B. Slight pressure is applied for 10 s to ensure that the new film stays in place, then water flow is immediately turned on for 2 hrs to examine results of healing. As shown in FIG. 34C, water is able to flow through the healed pipe without leakage. (In this example, R1 protein has amino acid sequence identical to SEQ. ID. No. 3, and R2 protein has amino acid sequence identical to SEQ. ID. No. 4.)

Detailed Description of the Invention

The compositions of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any of the additional or optional ingredients, components, or limitations described herein.

As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a” cell includes a plurality of cells, including mixtures thereof.

“About” in the context of amount values refers to an average deviation of maximum ±20%, ±10% or ±5% based on the indicated value. For example, an amount of about 500 kDa molecular weight refers to 500 kDa±100 kDa, 500 kDa±50 kDa or 500 kDa±25 kDa molecular weight.

An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.

The terms “spider silk proteins,” and “spidroins” are used interchangeably herein, referring to both native and recombinant proteins. The term “major ampullate spidroins” (MaSps) refers to a specific subtype of spidroins, whose native forms are often found in spider dragline silk. MaSps generally comprise a repetitive region optionally flanked by N-terminal domains (NTDs) and C-tcrminal domains (CTDs).

The terms “purification,” “separation,” and “isolation” are used interchangeably herein in connection to proteins, specifically referring to the separation of target proteins from other cellular materials, chemical substances, and/or impurities present in the mixture.

A “polynucleotide,” “nucleic acid,” or “nucleotide sequence” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide), but is preferably either single or double stranded DNA sequences. The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double- stranded polynucleotides. The terms “polynucleotide sequence” and “nucleotide sequence” are also used interchangeably herein.

An “amino acid sequence,” “peptide,” “polypeptide,” or “protein” is a sequence of amino acids (including both naturally occurring and non-naturally occurring amino acids), which may be found in native proteins or may be artificially engineered as recombinant proteins or parts thereof. The term should also be understood to include, as equivalents, polypeptides with additional modifications, including but not limited to phosphorylation, glycosylation, lipidation, etc.

A “coding sequence” or a sequence which “encodes” a particular protein, is a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence arc determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3’ to the coding sequence.

As used herein, the term “gene” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. As used herein, the term “recombinant protein” or “engineered protein” refers to a protein translated from non-naturally occurring nucleotide sequences, which may comprise artificially designed and engineered sequences, and may further comprise some naturally occuning sequences in combination with artificial sequences.

As used herein, “sequence identity” and “% identity,” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions as compared to the reference sequence for optimal alignment of the two sequences. The number of positions at which identical amino acid residues occur in both sequences is determined, yielding the number of matched positions, which is divided by the total number of positions in the window of comparison and the result multiplied by 100 to yield the percentage of sequence identity. The comparison window is the entire length of the sequence being referred to unless indicated otherwise.

As used herein, “% similarity” is calculated as described for “% identity,” with the exception that the hydrophobic residues Ala, Vai, Phe, Pro, Leu, He, Trp, Met, and Cys are similar; the basic residues Lys, Arg, and His are similar; the acidic residues Glu and Asp are similar; and the hydrophilic, uncharged residues Gin, Asn, Ser, Thr, and Tyr are similar. The remaining natural amino acid Gly is not similar to any other amino acid in this context.

The present invention provides a recombinant spider silk protein (rSSP) comprising a repeating REP domain wherein the REP domain comprises a plurality of repeating units and wherein each repeating unit comprises either R1 protein or R2 protein. In an embodiment, the recombinant spider silk protein of the present invention comprises a repeating REP domain wherein the REP domain comprises a plurality of repeating units and wherein each repeating unit consists of either R1 protein or R2 protein. In an embodiment, the recombinant spider silk protein of the present invention comprises a repeating REP domain wherein the REP domain consists of a plurality of repeating units and wherein each repeating unit consists of either R1 protein or R2 protein. Any embodiment of the recombinant spider silk protein of the present invention further comprises a N-terminal domain (NTD) and a non-repeating C-terminal domain (CTD) flanking the REP domain. Any embodiment of the recombinant spider silk protein of the present invention further consists of a N-terminal domain (NTD) and a non-repeating C-terminal domain (CTD) flanking the REP domain.

In an embodiment, the NTD of the recombinant spider silk protein of the present invention is derived from the NTD of major ampullate spidroins (MaSps) of Nephila pilipes. In an embodiment, the NTD of the recombinant spider silk protein of the present invention consists of amino acid sequence that is identical to amino acid sequence SEQ ID NO. 1. In an embodiment, the NTD of the recombinant spider silk protein of the present invention comprises amino acid sequence that is identical to amino acid sequence SEQ ID NO. 1. In yet another embodiment, the NTD of the recombinant spider silk protein of the present invention consists of an amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, and most preferably at least 95% identical to SEQ ID NO. 1. In yet another embodiment, the NTD of the recombinant spider silk protein of the present invention comprises amino acid sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, and most preferably at least 95% identical to SEQ ID NO. 1. In another embodiment, the NTD of the recombinant spider silk protein of the present invention comprises an amino acid sequence that is at least 80% similar, preferably at least 85% similar, more preferably at least 90% similar, and most preferably at least 95% similar to SEQ ID NO. 1. In yet another embodiment, the NTD of the recombinant spider silk protein of the present invention consists of an amino acid sequence that is at least 80% similar, preferably at least 85% similar, more preferably at least 90% similar’, and most preferably at least 95% similar to SEQ ID NO. 1.

SEQ ID NO. 1: [QNTPWSSTALADAFINAFLNEAGRTGAFTADQLDDMSTIGDTLKGAMDKMARSNKSS KSKLQALNMAFASSMAEIAAVEQGGMGVEAKTNAIADSLNAAFMQTTGSINSQFVNEI RSLISMFAQASANEV]

In an embodiment, the C-terminal domain (CTD) of the recombinant spider silk protein of the present invention is derived from the CTD of MaSps of Nephila pilipes. In another embodiment, the CTD of the recombinant spider silk protein of the present invention consists of amino acid sequence identical to the amino acid sequence of SEQ ID NO. 2. In another embodiment, the CTD of the recombinant spider silk protein of the present invention comprises amino acid sequence identical to SEQ ID NO. 2. In yet another embodiment, the CTD of the novel recombinant spider silk protein of the present invention consists of amino acid sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, and most preferably at least 95% identical to SEQ ID NO. 2. In yet another embodiment, the CTD of the novel recombinant spider silk protein of the present invention comprises amino acid sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, and most preferably at least 95% identical to SEQ ID NO. 2. In yet another embodiment, the CTD of the novel recombinant spider silk protein of the present invention comprises an amino acid sequence at least 80% similar, preferably at least 85% similar, more preferably at least 90% similar, and most preferably at least 95% similar to SEQ ID NO. 2. In another embodiment, the CTD of the novel recombinant spider silk protein of the present invention consists of an amino acid sequence that is at least 80% similar, preferably at least 85% similar, more preferably at least 90% similar, and most preferably at least 95% similar to SEQ ID NO. 2.

SEQ ID NO. 2: [GASAAASRLSSPEASSRVSSAVSNLVSSGPTNSAALSNTISNVVSQISSSNPGLSGCDVLV QALLEVVSALIHILGSSSIGQVNYGSAGQATQIV]

In an embodiment, NTD of the present invention forms a dipolar structure. In an embodiment, CTD of the present invention comprises cysteine, which can form a disulfide bond with the cysteine residue of another molecule. The properties of NTD and CTD of the present invention in the recombinant spider silk protein of the present invention facilitate aggregation and elongation during spinning.

In an embodiment, the R1 protein of the present invention is a repeat unit consisting of amino acid sequence identical to amino acid sequence of SEQ ID NO. 3. In an embodiment, the R1 protein of the present invention is a repeat unit comprising amino acid sequence identical to amino acid sequence of SEQ ID NO. 3. In an embodiment, the R1 protein of the present invention is a repeat unit comprising amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical or at least 95% identical to amino acid sequence of SEQ ID NO. 3. In an embodiment, the R1 protein of the present invention is a repeat unit consisting of amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical or at least 95% identical to amino acid sequence of SEQ ID NO. 3. In an embodiment, the R1 protein of the present invention is a repeat unit comprising amino acid sequence at least 70% similar, at least 75% similar, at least 80% similar, at least 85% similar, at least 90% similar or at least 95% similar to amino acid sequence of SEQ ID NO. 3. In an embodiment, the R1 protein of the present invention is a repeat unit consisting of amino acid sequence at least 70% similar, at least 75% similar, at least 80% similar, at least 85% similar, at least 90% similar or at least 95% similar- to amino acid sequence of SEQ ID NO. 3.

SEQ ID NO. 3:

[GAGAAAAAASGAGQGGYGRQGGQ]

In an embodiment, the R2 protein of the present invention is a repeat unit consisting of amino acid sequence identical to amino acid sequence of SEQ ID NO. 4. In an embodiment, the R2 protein of the present invention is a repeat unit comprising amino acid sequence identical to amino acid sequence of SEQ ID NO. 4. In an embodiment, the R2 protein of the present invention is a repeat unit comprising amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical or at least 95% identical to amino acid sequence of SEQ ID NO. 4. In an embodiment, the R2 protein of the present invention is a repeat unit consisting of amino acid sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical or at least 95% identical to amino acid sequence of SEQ ID NO. 4. In an embodiment, the R2 protein of the present invention is a repeat unit comprising amino acid sequence at least 70% similar, at least 75% similar, at least 80% similar, at least 85% similar, at least 90% similar or at least 95% similar to amino acid sequence of SEQ ID NO. 4. In an embodiment, the R2 protein of the present invention is a repeat unit consisting of amino acid sequence at least 70% similar, at least 75% similar-, at least 80% similar, at least 85% similar, at least 90% similar or at least 95% similar to amino acid sequence of SEQ ID NO. 4.

SEQ ID NO. 4:

[GPGGYGPGQQGPSGPGSAAAAAAAAGPGGYGPGQQ] Importantly, the recombinant spider silk proteins of the present invention possess self- healing property as illustrated in Examples 8-14 in connection with FIGs. 14A-14B, 17A-22B, 28-34. Properties of the recombinant spider silk proteins of the present invention such as the self-healing property is due at least partly to amino acid sequences of R1 and R2 wherein R1 comprises protein structures rich in crystalline P-sheets comprised of poly-A and/or poly-GA amino acid sequences and R2 comprises protein structures rich in beta- turn and/or beta- spiral comprised of GPGXX and/or 3i-helix comprised of GGX. The R1 repeat unit based upon SEQ ID NO. 3 is richer in crystalline P-sheets (SEQ ID NO. 3: beta-sheet crystalline: :48%, beta-turn: 0%, helix/coil:52%), lending rigidity property to the resulting silk whereas the R2 repeat unit based upon SEQ ID NO. 4 is richer in P-tum spirals (SEQ ID NO. 4: beta-sheet crystalline: 29%, beta-turn: 45%, helix/coil: 26%), lending elastic and self-healing properties to the resulting silk.

In an embodiment, the REP domain of the recombinant spider silk protein of the present invention comprises repeated units of R1 and R2 in different ratios to produce materials with desired strength, tensile properties, and self-healing properties as shown in Examples 15 to 21 in connection with FIGs. 23-34.

As shown in Examples 19 in connection with FIGs. 29 A and 29 B, films made with recombinant spider silk protein with REP domain consisting of R1 and R2 protein with R1:R2 ratio of between about 3: 1 to about 0: 1 provided higher healability at with tensile strength between about 24 to about 30 N/mm² and healing percentage above 65%. In addition, as shown in Examples 15 and 17 in connection with FIGs. 24 and 26, films made with recombinant spider silk protein with REP domain consisting of one or more of R1 and one or more of R2 protein with R1:R2 ratio of between about 1:0 to about 1:3 provided high weight retention after water treatment, with R1:R2 ratio of 1:9 having about 50% weight retention and R1:R2 ratio of 0: 1 completely dissolving.

Therefore, in an embodiment, the REP domain of the spider silk protein of the present invention comprises one or more of R1 proteins, one or more of R2 proteins or a combination thereof. In an embodiment, the recombinant spider silk protein of the present invention consists of both one or more of R1 and one or more of R2 proteins. In another embodiment, the recombinant spider silk protein of the present invention comprises both one or more of R1 and one or more of R2 proteins. In an embodiment, the REP domain of the recombinant spider silk protein of the present invention comprises one or more of R1 proteins and one or more of R2 proteins in a ratio in terms of numbers of R1 and to numbers of R2 of about 1 : 10 to about 10: 1 such as about 1: 10, 1:9, 1:8, 1:7, 1:6. 1:5, 1:4. 1:3, 1:2, 1: 1, 2: 1, 3:1, 4: 1, 5: 1. 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1 including any ratio or ratio ranges falling within these values. In an embodiment, the REP domain of the recombinant spider silk protein of the present invention comprises one or more of R1 proteins and one or more of R2 proteins in a ratio in terms of numbers of R1 and to numbers of R2 proteins of about 3: 1 to about 1:5 such as about 3: 1, 2:1, 1: 1, 1:2, 1:3, 1:4 or 1:5 including any ratio or ratio ranges falling within these values. In an embodiment, the REP domain of the recombinant spider silk protein of the present invention comprises one or more of R1 proteins and one or more of R2 proteins in a ratio in terms of numbers of R1 and to numbers of R2 of about 3: 1 to about 1:5 such as about 3: 1 to about 1:5 or about 1: 1 to about 1:5 or about 1: 1 to about 1:3.

In an embodiment, the one or more of R1 protein and the one or more of R2 protein are about uniformly distributed in the recombinant spider silk protein of the present invention. In an embodiment, the one or more of R 1 protein and the one or more of R2 protein are randomly distributed in the recombinant spider silk protein of the present invention. In an embodiment, the one or more of R1 protein and the one or more of R2 protein are about evenly blended within the recombinant spider silk protein of the present invention.

In an embodiment, the REP domain of the recombinant spider silk protein of the present invention comprises ratio of one or more of the beta sheet structures to one or more of the amorphous structures of about 75:25 to about 45:55 in terms of number of structures wherein the amorphous structure comprises beta-turn and/or beta-spiral comprised of GPGXX and/or 3i- helix comprised of GGX. In an embodiment, the REP domain of the recombinant spider silk protein of the present invention comprises ratio of one or more of the beta sheet structures to one or more of the amorphous structures of about 60:40 to about 45:55. In an embodiment, the REP domain of the recombinant spider silk protein of the present invention comprises ratio of one or more of the beta sheet structures to one or more of the amorphous structures of about 60:40 to about 50:50.

In an embodiment, one or more of the beta sheet crystalline structures and one or more of the amorphous structures are about uniformly distributed in the recombinant spider silk protein of the present invention. In an embodiment, one or more of the beta sheet crystalline structure and one or more of the amorphous regions arc randomly distributed in the recombinant spider silk protein of the present invention. In an embodiment, one or more of the beta sheet crystalline structures and one or more of the amorphous structures are about evenly blended within the recombinant spider silk protein of the present invention.

In an embodiment, the REP domain of the spider silk protein of the present invention comprises one or more of the R1 proteins and no R2 proteins. In an embodiment, the REP domain of the spider silk protein of the present invention consists of one or more of the R1 proteins and no R2 proteins. In an embodiment, the REP domain of the spider silk protein of the present invention comprises one or more of the R1 proteins and no effective amount of R2 proteins. In another embodiment, the REP domain of the spider silk protein of the present invention consists of one or more of the R2 proteins and no R1 proteins. In another embodiment, the REP domain of the spider silk protein of the present invention comprises one or more of the R2 proteins and no R1 proteins. In another embodiment, the REP domain of the spider silk protein of the present invention comprises one or more of the R2 proteins and no effective amount of R1 proteins.

In an embodiment, the recombinant spider silk protein of the present invention comprises about 4 to about 300 repeat units such as about 4, about 8, about 20, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275 or about 300 repeat units including any number and number ranges falling within these values. In an embodiment, the novel recombinant spider silk protein of the present invention comprises about 8, about 16, about 32, about 64, about 96, or about 128 repeat units. In yet another embodiment, the novel recombinant spider silk protein of the present invention comprises up to 144 repeat units.

In an embodiment, any embodiment of the recombinant spider silk protein of the present invention has a molecular weight of from about 5 to 600 kDa, from about 10 to 500 kDa, from about 20 to 400 kDa, or over 800 kDa. In an embodiment, any embodiment of the recombinant spider silk protein of the present invention has a molecular weight of from about 5 to 600 kDa such as about 5, about 30, about 60, about 90, from about 100, about 130, about 160, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550 or about 600 kDa including any molecular weight or molecular weight ranges falling within these values. Tn an embodiment, any embodiment of the recombinant spider silk protein of the present invention has a molecular weight of over about 60 kDa. In an embodiment, any embodiment of the recombinant spider silk protein of the present invention may be linked with mEGFP and/or mCherry protein(s). In another embodiment, any embodiment of the recombinant spider silk protein is tag-free. In an embodiment, other functional proteins may be attached to the NTD or CTD of any embodiment of the recombinant spider silk protein of the present invention.

In an embodiment, the recombinant spider silk protein of the present invention may exhibit toughness of up to about 190, 200, 210, 220, 230, 240, or 250 MJ/m³, comparable to that of native spider silk as demonstrated in Example 3 and FIGs. 3-5 below. In an embodiment, the recombinant spider silk protein of the present invention may also exhibit self-healing properties.

In an embodiment, the recombinant spider silk protein comprises a hydrophilic NTD and CTD as well as a hydrophobic REP domain. In an embodiment, this hydrophilic -hydrophobic- hydrophilic arrangement allows the recombinant silk proteins of the present invention to form micelle-like structures. Without being bound to theory, these micelle-like structures facilitate purification of the recombinant spider silk protein of the present invention in a tag-free and timesaving manner using the purification method of the present invention as described in further detail below in connection with Examples 2, 5 and 6 in connection with FIGs. 2A-2D, 6A- 8B,10A-10C and 12A-12D.

In another embodiment of the present invention, threads, films, patches, fabrics, textiles, medical devices, threads, and other products produced using the recombinant spider silk protein of the present invention may also comprise additional materials such as graphene. Other materials that may be blended with the recombinant spider silk protein of the present invention include but are not limited to: graphite, carbon nanotubes, metal oxides such as titanium dioxide, iron (II) oxide, magnetite (Fe3O4), copper oxide, gold oxide, and silver oxide, ceramics such as silicon oxide, and other synthetic nanoparticles such as quantum dots, etc.

The present invention also provides a method of purifying any embodiment of the recombinant spider silk protein of the present invention. An embodiment of the purification method of the present invention is illustrated in FIG. 15 which begins with step 210 of lysing prokaryotic or eukaryotic system using a lysis buffer wherein the prokaryotic or eukaryotic system is capable of expressing the novel recombinant spider silk protein of the present invention. In an embodiment, the prokaryotic or eukaryotic system capable of expressing the novel recombinant spider silk protein of the present invention comprises bacteria, yeast, mammalian cells, plants, insect cells and transgenic animal cells or, more specifically, E. coli cells. In step 220, the resulting lysis solution is centrifuged to obtain cell pellet. In step 230, the cell pellet of step 220 is resuspended in lysis buffer and sonicated on ice, then centrifuging the solution to obtain cell pellet. In step 240, the cell pellet of step 230 is resuspended using sodium dodecyl sulfate (SDS) buffer, then sonicated the resulting solution on ice and centrifuging the solution to obtain the supernatant. In step 250, the supernatant of step 240 is centrifuged to obtain recombinant spider silk protein of the present invention. Next in step 260, the recombinant spider silk protein of the present invention of step 240 is washed using DI water or ammonium bicarbonate buffer several times. In an embodiment, steps 220 and 230 may be repeated as desired.

In an embodiment, the process of step 210 and 220 takes about 40 minutes to about 80 minutes such as about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes or about 80 minutes including any minutes and minute ranges that fall within these values; step 230 takes about 40 minutes to about 80 minutes such as about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes or about 80 minutes including any minutes and minute that fall within these values; step 240 takes about 5 minutes to about 30 minutes such as about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, or about 30 minutes including any minutes and minute that fall within these values; step 250 takes about 20 minutes to about 40 minutes such as about 20 minutes, about 30 minutes or about 40 minutes including any minutes and minute falling within these values; and step 260 takes about 40 minutes to about 80 minutes such as about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes or about 80 minutes including any minutes and minute that fall within these values. In another embodiment, the process of step 210 and 220 takes about 1 hr, step 230 takes about 1 hr, step 240 takes about 0.5 hr, step 250 takes about 30 min, and step 260 takes about 1 hr.

In an embodiment, the recombinant spider silk protein of the present invention may be purified in a tag-free process. In another embodiment, the method for purifying recombinant spider silk protein of the present invention does not include the use of affinity or diffusion-based methods such as columns, chromatography, dialysis and the like, contributing to the method’s substantially time-saving manner. Remarkably, purification methods commonly used by those skilled in the ail involving the use of affinity or diffusion-based processes may take up to or more than 2 days, while the purification method of the present invention may be completed in 1 day, and more preferably in under 12 hours. In an embodiment, the purification method of the present invention results in a composition comprising about 80%. about 85%, about 90%, about 95%, about 98%, about 99% or about 99.9% of one or more embodiments of the recombinant protein of the present invention measured using absorption test based purity measurement method of Example 4.

In an embodiment of the present invention, the lysis buffer used in the method disclosed herein comprises about 0.1 to 0.5 mg/mL lysozyme, about 0.1 to 1.0 wt% Triton X-100, and about 10 to 100 mM ammonium phosphate. Preferably, the lysis buffer comprises about 0.2 to 0.3 mg/mL lysozyme, about 0.3 to 0.7 wt% Triton X-100, and about 30 to 70 mM ammonium phosphate. In another embodiment, the lysis buffer comprises about 0.25 mg/mL lysozyme, about 0.5 wt% Triton X-100, and about 50 mM ammonium phosphate. In an embodiment, the lysis buffer of the present invention has a pH level of from about 7.5 to 8.5 or about 8.0.

In an embodiment of the present invention, the SDS buffer used in the method disclosed herein comprises about 1 to 20 wt% sodium dodecyl sulfate, about 0.1 to 1.0 wt% Triton X-100, and about 1 to 20 mM Tris-Cl. Preferably, the SDS buffer comprises about 5 to 15 wt% sodium dodecyl sulfate, about 0.3 to 0.7 wt% Triton X-100, and about 5 to 15 mM Tris-Cl. More preferably, the SDS buffer comprises about 10 wt% sodium dodecyl sulfate, about 0.5 wt% Triton X-100, and about 10 mM Tris-Cl.

An embodiment of the present invention comprises a recombinant spider silk protein which may be expressed using cell-free in vitro methods such as cell-free protein synthesis as disclosed in Example 6 below, as well as in vivo methods of expression using prokaryotic or eukaryotic systems. In vivo expression methods may involve recombinant expression systems using a variety of suitable hosts, including but not limited to bacteria, yeast, mammalian cells, plants, insect cells and transgenic animals. In an embodiment, the novel recombinant spider silk protein is produced using Escherichia coli cells. In an embodiment of the present invention, the purification method disclosed herein produces proteins of up to 85%, 90%, 95%, 98%, 99% or 100% purity wherein purity is determined based upon absorption test disclosed in Example 4.

In an embodiment of the present invention, the purified novel recombinant spider silk protein resulting from the purification method disclosed herein is not denatured. In another embodiment, the resulting recombinant spider silk protein of the present invention spontaneously forms micelle-like structures.

The present invention further comprises any nucleotide sequences encoding any of the novel recombinant spider silk protein of the present invention disclosed above. The present invention also comprises a vector containing a nucleotide sequence encoding any of the novel recombinant spider silk protein of the present invention disclosed above.

It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In general, the terms used in the disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology.

Examples:

Example 1: Production of novel recombinant spider silk protein using E. coli

E. coli cells engineered to express a novel recombinant spider silk protein of the present invention of NT-REP-CT were cultivated in a 5 L bioreactor (FS-01-A05, Major Science) using 2 L Terrific Broth as culture medium. Phosphate salt and the culture medium were sterilized separately under high heat and pressure (121°C, 1.5 atm, 15 min) then allowed to cool and filled into the bioreactor. The culture medium is adjusted to pH 7.0 using 4 N NaOH and 10% phosphoric acid (stir rate 400-800 rmp, ventilation 3 vvm). 20 mL engineered E. coli cells (1% v/v of the medium) were inoculated in the bioreactor. 25 pg/mL kanamycin, 12.5 pg/mL chloramphenicol, and 50 pg/mL ampicillin, antibiotics corresponding to antibiotic-resistant genes present on the plasmid transferred into the engineered E. coli cells, were added to the culture medium. The cells were cultured at 30°C and pH-stat control strategy was applied to maintain a pH level of 7.0 when glycerol was depleted. When the ODeoo of the culture reached 30, the culture temperature was adjusted to 20°C, and Isopropyl P-D-l-thiogalactopyranosidc (IPTG) was added to the tank for a final concentration of 0.5 mM. IPTG induction lasted for 24 to 30 hrs.

FIG. 1A-1D show transmission electron microscope (TEM) images of the recombinant spider silk proteins NT-MaSpl*32-CT-mEGFP and NT-MaSp2*32-CT-mCherry expressed in engineered E. coli cells. NT-MaSpl*32-CT-mEGFP is a novel recombinant spider silk protein of the present invention with an NTD comprising the amino acid sequence of SEQ ID NO. 1, a CTD comprising the amino acid sequence of SEQ ID NO. 2, a REP domain comprising 32 repetitions of SEQ ID NO. 3, and an mEGFP protein linked to the C-terminus. NT-MaSp2*32- CT-mCherry is another novel recombinant spider silk protein of the present invention with an NTD comprising the amino acid sequence of SEQ ID NO. 1, a CTD comprising the amino acid sequence of SEQ ID NO. 2, a REP domain comprising 32 repetitions of SEQ ID NO. 4, and an mCherry protein linked to the C-terminus. The fluorescence genes mEGFP and mCherry were linked onto target recombinant silk proteins for ease of observation of the presence of NT- MaSpl*32-CT-mEGFP and NT-MaSp2*32-CT-mCherry. In FIG. 1A, NT-MaSpl*32-CT- mEGFP is shown in green. In FIG. IB, NT-MaSp2*32-CT-mCherry is shown in red. FIG. 1C is an overlay image of FIG. 1A and IB, demonstrating that both recombinant proteins were produced in the same bacterial cells. FIG. ID is a bright field image of the individual E. coli cells. It is apparent from these four images that the recombinant proteins NT-MaSpl*32-CT- mEGFP and NT-MaSp2*32-CT-mCherry were expressed simultaneously in the same individual E. coli cells.

Example 2: A tag-free and time-saving purification method of recombinant spider silk protein

10 g of E. coli cells engineered to express a novel recombinant spider silk protein of the present invention were first lysed using 50 mL lysis buffer comprising 0.25 mg/mL lysozyme, 0.5 wt% Triton X-100, and 50 mM ammonium phosphate adjusted to pH 8.0. The resulting solution was then centrifuged at 10,000 xg for 30 min to obtain the pellet. The pellet was resuspended using 25 mL lysis buffer and sonicated on ice for 30 min. Following sonication, the resulting solution was centrifuged again at 10,000 xg for 30 min to obtain the pellet. The pellet was resuspended using 25 mL SDS buffer comprising 10 wt% sodium dodecyl sulfate, 0.5 wt% Triton X-100, and 10 mM Tris-Cl adjusted to pH 8.0, then sonicated on ice for 30 min to obtain the supernatant. The supernatant was centrifuged at 1,000 xg for 10-15 min to obtain the recombinant spider silk protein of the present invention. The recombinant spider silk protein of the present invention was washed using deionized (DI) water or 10 mM ammonium bicarbonate buffer 3-5 times, then stored in a 4°C refrigerator or lyophilized for long-term storage. SDS- PAGE analyses of FIG. 16A and 16B show that purified recombinant spider silk proteins have purity of up to or above 98%.

FIG. 2A-2D show transmission electron microscope (TEM) images of the recombinant spider silk proteins NT-MaSpl*32-CT-mEGFP and NT-MaSp2*32-CT-mCherry purified from engineered E. coli cells. In FIG. 2A, NT-MaSpl*32-CT-mEGFP is shown in green. In FIG. 2B, NT-MaSp2*32-CT-mCherry is shown in red. FIG. 2C is an overlay image of FIG. 2A and 2B, demonstrating the presence of both recombinant proteins in the same micelle-like structures. FIG. 2D is a bright field image of the purified recombinant proteins. It is apparent from these four images that the recombinant proteins NT-MaSpl*32-CT-mEGFP and NT-MaSp2*32-CT- mCherry were co-localized in micelle-like structures and remained so after purification.

Example 3: Mechanical properties of the novel recombinant spider silk proteins

Purified recombinant spider silk protein NT-MaSpl *32-CT of the present invention, comprising an NTD comprised of SEQ NO. 1, a REP domain comprised of 32 repetitions of SEQ NO. 3, and a CTD comprised of SEQ NO. 2, was lyophilized, then dissolved using 20 w/v% hexafluoroisopropanol (HFIP). Protein samples were loaded into a 26 gauge syringe and injected slowly into 95% ethanol, then stretched in 75% isopropanol. The samples were then dried and fixed for use in subsequent mechanical property tests.

Silk samples were fixed onto paper using tape, leaving a gauge of 1 cm. Microscopes were used to measure the diameter of the silk samples. The samples were placed onto a tensile tester (SHIMADZU EZ-SX), held in place with clamps at both the top and the bottom, ensuring that samples naturally hung in straight, even lines. Samples were cut from both ends along the dotted line, leaving only silk samples connecting both ends of the clamps. Stretching was performed at a rate of 1 mm/min, during which the tensile tester automatically recorded data until silk samples broke. Data were then graphed into a stress-strain graph. FIG. 3 A shows a sample of NT-MaSpl *32-CT stretched and molded for mechanical testing. The thickness of the silk was about 10.67 pm. FIG. 3B is a graph demonstrating the toughness of NT-MaSpl*32-CT, with true strain in percentages as the x-axis and true stress (also known as true strength) in MPa as the y-axis. The definitions of stress and strain are as commonly known to those skilled in the art. The red, green, and blue lines represent three replicated tests. The tensile toughness of NT-MaSpl*32-CT, calculated from the area under the average stress-strain curve, is 38.63 ± 15.93 MJ/m³.

Purified recombinant spider silk protein NT-MaSp2*32-CT of the present invention, comprising an NTD comprised of SEQ NO. 1, a REP domain comprised of 32 repetitions of SEQ NO. 4, and a CTD comprised of SEQ NO. 2, was combined with hexafluoroisopropanol (HFIP) so that the final concentration of HFIP is about 25-33 v/v% and the resulting protein sample becomes more aggregated, clear, and sticky. The sample was centrifuged and collected, then loaded into a 26 gauge syringe and slowly injected into a 500 mM ammonium phosphate coagulation bath (pH 5.0). After resting for a few minutes, the sample was stretched in 75% isopropanol then soaked in 95% ethanol. The sample was then dried and fixed for use in subsequent mechanical property tests as described above.

FIG. 4A shows a sample of NT-MaSp2*32-CT stretched and molded for mechanical testing. The thickness of the silk sample is about 13.83 pm. FIG. 4B is a graph demonstrating the toughness of NT-MaSp2*32-CT, with true strain in percentages as the x-axis and true strength in MPa as the y-axis. The red, green, and blue lines represent three replicated tests. The tensile toughness of NT-MaSp2*32-CT, calculated from the area under the average stress-strain curve, is 63.53 ± 10.91 MJ/m³.

Purified recombinant spider silk protein NT-MaSp2*96-CT of the present invention, comprising an NTD comprised of SEQ NO. 1, a REP domain comprised of 96 repetitions of SEQ NO. 4, and a CTD comprised of SEQ NO. 2, was combined with hexafluoroisopropanol so that the final concentration of HFIP is about 25-33 v/v% and the resulting protein sample becomes more aggregated, clear, and sticky. The sample was centrifuged and collected, then loaded into a 26 gauge syringe and slowly injected into a 500 mM ammonium phosphate coagulation bath (pH 5.0). After resting for a few minutes, the sample was stretched in 75% isopropanol then soaked in 95% ethanol. The sample was then dried and fixed for use in subsequent mechanical property tests as described above.

FIG. 5A shows a sample of NT-MaSp2*96-CT stretched and molded for mechanical testing. The thickness of the silk sample is about 14.05 pm. FIG. 5B is a graph demonstrating the toughness of NT-MaSp2*96-CT, with true strain in percentages as the x-axis and true strength in MPa as the y-axis. The red, green, and blue lines represent three replicated tests. The tensile toughness of NT-MaSp2*96-CT, calculated from the area under the average stress-strain curve, is 180.35 ± 7.45 MJ/m³, similar to that of native spider silks.

Example 4: Absorption tests Following purification, novel recombinant spider silk proteins of the present invention was dissolved in 5 M guanidine thiocyanate, then sample absorption at 260 nm and 280 nm was measured, with guanidine thiocyanate of the same concentration serving as blank. The A260/A280 ratio was calculated. A ratio of 0.5 to 0.6 would indicate high protein purity with minimal nucleic acid contamination.

Table 1

Table 1 shows the A260/A280 test results of NT-MaSpl*32-CT.

Table 2

Table 2 shows the A260/A280 test results of NT-MaSp2*32-CT.

Table 3 Table 3 shows the A260/A280 test results of NT-MaSp2*96-CT.

As shown in Tables 1 to 3, the A260/A280 ratios of purified NT-MaSpl*32-CT, NT- MaSp2*32-CT, and NT-MaSp2*96-CT all fall between 0.5 and 0.6, indicating high protein purity with minimal nucleic acid contamination. It should be noted that A260/A280 ratio of 0.57 indicates 100% protein purity with no nucleic acid contamination.¹

Example 5: Transmission electron microscopy (TEM)

To prepare negatively stained samples for observation under a transmission electron microscope, 10 pL protein samples of 3-5 wt% recombinant protein sample suspended in 10 mM Tris-Cl at pH 8.0 were loaded onto carbon film supported copper grids (01800-F; TED PELLA) and left at room temperature for 1 min. Dry filter papers were used to absorb excess liquid. The loaded films were washed 3 times using deionized and distilled water, then stained 3 times using 10 pL 2% uranyl acetate (Merck), wherein the dyeing intervals were 10 s, 10 s, and 1.5 min, respectively. After excess dyes were absorbed, the films were dried in a drying oven overnight. A transmission electron microscope (H-7100; Hitachi) was used to observe the protein samples. FIG. 6 to 8 show that micelles formed from the sample proteins of the instant invention had sizes ranging from 200 to 1 ,000 nm.

FIG. 6 shows negatively stained TEM images of the recombinant spider silk protein NT- MaSpl*32-CT in the form of micelles. The approximate micelle size of NT-MaSpl*32-CT is about 150 to 600 nm.

FIG. 7 shows negatively stained TEM images of the recombinant spider silk protein NT- MaSp2*32-CT in the form of micelles. The approximate micelle size of NT-MaSp2*32-CT is about 150 to 600 nm.

FIG. 8 shows negatively stained TEM images of the recombinant spider silk protein NT- MaSp2*96-CT in the form of micelles. The approximate micelle size of NT-MaSp2*96-CT is about 150 to 1000 nm.

Example 6: Cell-free protein synthesis

¹ Glasel JA. Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18(1), 62-3 (1995). Cell-free protein synthesis (CFPS) was used for the observation of the novel recombinant spider silk protein in a cell-free system. In order to avoid structural alterations of the NTD brought on by the pH levels of CFPS reactions, point mutations D35K and K60D were performed in the NTD amino acid sequence. The mutated NT domain is hereafter denoted as NTCR. and the unaltered NT domain is denoted as NT. NT-MaSpl*32-CT with mEGFP attached (a recombinant spider silk protein with an NTD and CTD of the present invention, 32 repeats of SEQ ID NO. 3 as the REP domain, and mEGFP attached for observation using fluorescence microscopy, hereafter NT-MaSpl*32-CT-mEGFP), NTcR-MaSpl*32-CT with mEGFP attached (a recombinant spider silk protein with NTD modified at D35K and K60D, a CTD of the present invention, 32 repeats of SEQ NO. 3 as the REP domain, and mEGFP attached for observation using fluorescence microscopy, hereafter NTcR-MaSpl*32-CT-mEGFP, and NTcR-MaSp2*32- CT with mCherry attached (a recombinant spider silk protein with NTD modified as described above, a CTD of the present invention, 32 repeats of SEQ ID NO. 4 as the REP domain, and mCherry attached for observation using fluorescence microscopy, hereafter NTcR-MaSp2*32- CT-mCherry) were used in cell-free protein synthesis.

NTD structural stability test

40 mM MES or 40 mM Tris was prepared with pH adjusted to 6.0 to 8.8, with intervals of 0.4 between each buffer. NT and NTCR proteins purified using His tag and dialysis were diluted with the aforementioned buffers to 10 pM. Samples were loaded into a 96 well black/clear bottom plate (IsoPlate; PerkinElmer) for fluorescence measurements. With absorption at 280 nm, sample fluorescence emission was measured from 300 nm to 400 nm with intervals of 1 nm. The ratio of 339 nm/351 nm fluorescence emission, the emission of tryptophan (Trp) which is used to monitor changes in protein structure, at different pH levels was calculated and the results shown as a line graph in FIG. 9. The fluorescence ratio of recombinant spider silk protein with unaltered NT domain lowered from pH 7.6 to 7.2, indicating a structural change which exposed Trp residue in NTD, indicating structural instability. The fluorescence ratio of recombinant spider silk protein with mutated NTCR domain remained stable at different pH levels, indicating structural stability at different pH levels.

Preparation of 4x master mix Preparation of the 4x master mix was based on Cai et al.² with some changes made specifically for the present invention. The concentrations used in CFPS reactions arc listed in Table 4. L-Tyrosine was adjusted to pH 11 using KOH to increase solubility.

Table 4 Preparation of cell extract

Cell extract was prepared using E. coli BLR (DE3) according to Kwon and Jewett³.

Reaction conditions

25 pL 4x master mix, 30 pL cell extract, 50 pg/mL T7 RNA polymerase, and 10 pg/mL plasmid DNA with genes encoding recombinant spider silk proteins of the present invention were loaded into 1.5 mL Eppendorfs, and ddH O was added to adjust volume to 100 pL. Eppendorf lids were removed, and Nunc™ breathable sealing tapes were used to seal the Eppendorfs. Finally, the loaded Eppendorfs were centrifuged at 30°C and 250 rpm to react for 10 hrs.

² Cai, Q. et al. A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems. Biotechnol Prog 31(3). 823-831 (2015).

³ Yong-Chan Kwon and Michael C. Jewett. High-throughput preparation methods of crude extract for robust cell- free protein synthesis. Sci Rep 5, 8663 (2015). CFPS results

Fluorescence microscopy was used to observe the recombinant spider silk protein of the present invention expressed using a cell-free production system. FIGs. 10A-10C show fluorescence microscopy imaging of the CFPS results. FIG. 10A shows fluorescence microscopy imaging of the positive control group, CFPS used only to produce mEGFP. As demonstrated in the figure, the green fluorescence of mEGFP was evenly distributed. FIG. 10B shows fluorescence microscopy imaging of NT-MaSpl*32-CT-mEGFP. As demonstrated in the figure, green fluorescence, representing the recombinant protein of the present invention with mEGFP attached for ease of observation, was disordered and aggregated. FIG. 10C shows fluorescence microscopy imaging of NTcR-MaSpl*32-CT-mEGFP. As demonstrated in the figure, green fluorescence was present in a particulate, micelle-like form.

Dynamic light scattering (DLS) was used to analyze NTcR-MaSpl*32-CT-mEGFP produced using CFPS. FIG. 11 shows the result of DLS analysis with diameter in nm as the x- axis and frequency in percentages as the y-axis. The resulting Z-average or particle size was 146.5 nm, while the polydispersity index PI was 0.462.

Fluorescence microscopy was used to observe NTcR-MaSpl*32-CT-mEGFP and NTCR- MaSp2*32-CT-mCherry. FIGs. 12A-12D show fluorescence microscopy imaging of the recombinant spider silk proteins of the present invention, with NTcR-MaSpl *32-CT-mEGFP in green in FIG. 12A, NT_CR-MaSp2*32-CT-mCherry in red in FIG. 12B, FIG. 12A and 12B overlaid in FIG. 12C, and the bright field image in FIG. 12D. As demonstrated in the figures, the two recombinant spider silk proteins produced using CFPS still spontaneously assemble into the same micelle-like structures.

Example 7: Hexafluoroisopropanol (HFIP) film test

Preparation of HFIP film

3 cm x 1 cm molds were made using Teflon. HFIP was added to lyophilized recombinant spider silk protein samples of NT-MaSp2*96-CT, a recombinant spider silk protein with NTD and CTD of the present invention and 96 repeats of SEQ NO. 4 as the REP domain, to 50 mg/mL concentration. 300 pL protein sample was poured into the mold and air dried for at least 2 hrs to form a thin film. After removing the uneven edges and cutting into appropriate sizes, the films were soaked in 95% ethanol for 1 to 2 hrs. The films were then removed from ethanol, flatten with plastic discs, and air dried for 30 min.

Healing test

The protein film was cut in the middle, then the two halves placed together with about 0.3 cm overlapping. After addition of an adequate amount of water, an oil press was used to exert 1.3 MPa (will have to double check again) pressure upon the overlapping films for 15 min. After drying, the film was subjected to mechanical property tests using a tensile testing machine.

FIG. 13 shows the results of tensile testing with strain in percentages as the x-axis and stress in N/mm² as the y-axis. The black line represents the stress-strain curve of uncut NT- MaSp2*96-CT film and treated only with ethanol while the red line represents NT-MaSp2*96- CT cut, soaked in ethanol and had 1.3 MPa pressure exerted upon it. As demonstrated in the figure, healing of the recombinant spider silk film occurs with the aid of water and pressure, and can recover some mechanical properties. Notably, the location of fracture on the cut and healed film sample was different from the location of adhesion, which shows that the recombinant spider silk protein of the present invention exhibits self-healing properties.

Example 8: Self-healing test

Two fibers produced using the novel recombinant spider silk protein NT-MaSp2*96-CT of the present invention were wound together using a black string with hair clips used to hold both ends of the string. The fibers before healing are shown in FIG. 14A. The silk fibers were incubated in hot water at about 80 to 90°C for about 7 min, then air dried for about 45 min at ambient temperature. The string and hair clips were then removed. FIG. 14B shows the results of hot water treatment. As demonstrated in the photo, the two fibers have healed up into one.

To validate self-healing results of the fibers, the healed up fibers were used to lift hair clips. The healed up fiber could successfully hold up 1 hair clip of about 0.65 g, but was unsuccessful in holding up 3 hair clips.

Example 9: Healing comparison of different silks

Repairability of native spider silk films originating from different spider species was compared. Two species of spiders (orb- weaver Nephila pilipes and tent-builder Cyrtophora moluccensis) were selected and major-ampullate dragline silks were collected from the two species separately. Precast silk films from both spiders (2.5% w/v in HFIP) were cut using a razor blade to introduce rifts. The incision width of the cracked region on both silk films was monitored both before and after addition of water droplet. FIG. 17A shows change of incision width after healing process for both Nephila pilipes and Cyrtophora moluccensis major- ampullate dragline silk, indicating intrinsic material repairability of native spider silks. FIG. 17B shows the change of crack thickness as an index of spider silk film healing capacity. The width difference of spider silk films was normalized to its original gap thickness and plotted. Both FIG. 17A and 17B show that film produced using N. pilipes silk demonstrated superior healability to that of C. moluccensis.

Example 10: Healable and conductive spider silk-graphene films

Films containing recombinant spider silk protein of the present invention as well as 5%, 10%, and 15% graphene were produced as a potential bioelectronic scaffold. Film samples were cut and allowed to heal, with conductivity tests done before cutting and after healing. Uncut graphene spider silk films could conduct electricity, lighting up LED lights. Healed graphene spider silk films could also conduct electricity, lighting up LED lights to a lesser extent, as shown in FIG. 18.

Example 11 : Repairable spider silk films

Spider silk films produced using the recombinant spider silk protein NT-MaSp2*32-CT, a novel recombinant spider silk protein of the present invention with an NTD comprising the amino acid sequence of SEQ NO. 1, a CTD comprising the amino acid sequence of SEQ NO. 2, and a REP domain comprising 32 repetitions of SEQ NO. 3, were cut and healed, then subjected to mechanical tests. The healing method comprises the step of introducing 1-3 fl L of water to the films surface to wet the films surface and then contact the two pieces of the films together gently. Afterwards, the samples were air-dried on the bench for a few hours/or overnight to allow the water removal/evaporation. As demonstrated in FIG. 19, the cut and healed spider silk films exhibited comparable mechanical strength to that of the uncut films.

Example 12: Post treatments of cast silk materials and film repairability evaluation Spider silk films produced using NT-MaSp2*32-CT were cast in HFIP. After cutting, films were treated with 100% EtOH, film treated with 70% EtOH, film treated with 37°C water vapor annealing for 2 hrs, film treated with 60°C water vapor annealing for 1 hr, as well as film treated with 60°C water vapor annealing for 2 hrs, followed by film-healing challenges. The histogram of FIG. 20A demonstrates the healing capability of such films, showing the stress that can be endured by silk films post-treatment and after healing as compared to uncut films subjected to the same treatment. Black bars show the effect of post-treatments on mechanical strength of the films, while gray bars show the corresponding healing capability. Both 37°C (2 hrs) and 60°C (1 hr) treatments rendered exceptional mechanical performance after healing.

FIG. 20B graphs rehealing stress and strain of cast silk materials and film produced using the recombinant spider silk protein NT-MaSp2*32-CT. Films treated under 60°C water vapor annealing (1 hr) exhibited multiple-healing/reversible-healing capacities. The same treated films were repetitively healed and separated 1 to 5 times, and results show no significant changes in mechanical performance (the 5^th time resulted in slightly reduced but still good strength). Neither strength (shown in the histogram in blue) nor elasticity (shown in the histogram in red) were affected.

Example 13: Fabrication and application of treated silk materials

NT-MaSp2*32-CT films treated with 37°C water vapor annealing were woven and healed in various dimensional configurations, as shown in FIG. 21 A. Both silk film strips (the black being graphene doped silk samples, the transparent being pure silk samples) could form intertwined structures in ID, 2D, and 3D after the healing process.

FIG. 2 IB shows that fabricated silk strips (treated with 60°C water vapor annealing for 1 hr) were able to adhere, forming configurable rings with adjustable sizes. The rings were reopened and self-healed repetitively. The same silk-based ring was readjusted to fit either the thumb or the little finger, conferring a wearable device as a smart resistance sensor for sensing human fingers or skin. The device is set as a finger/skin recognition system using electrical resistance, turning “on” only when fingers are detected, and remaining “off’ when the resistance is too low or too high. Such a device may be used as part of a wearable biosensor or bioelectronic device. Example 14: Repairability of silk films produced using native spider silk and silkworm silk

The silks of orb-weaver spider Nephila pilipes, tent-builder spider Cyrtophora moluccensis. and mulberry silkworm Bombyx mori were used, with major-ampullate dragline silks collected from the two spiders and silk samples harvested from degummed cocoons of B. mori. The precast silk films from both spiders (2.5% w/v in HFIP) were cut by a razor blade to introduce rifts. The incision width of the cracked region on both silk films was measured before and after addition of water droplets to induce healing. FIG. 22A shows change of incision width after the healing process, demonstrating the intrinsic material repairability of silk materials harvested from the spiders and the silkworm. FIG. 22B shows the change of crack width as an index of silk film healing ability. The width difference of silk films was normalized to its original gap thickness and plotted using the formula shown in the figure and plotted. The silk film of N. pilipes showed superior healability to those of B. mori and C. moluccensis .

Example 15: Water resistance of recombinant spider silk protein

To investigate whether different ratios of R1 and R2 in films can affect water resistance stability, films of the following R1:R2 ratios are selected: 100:0, 75:25, 50:50, 33:67, 25:75, 10:90, and 0: 100, as shown in FIG. 23. Film created from pure R2 protein (0: 100) is used as a control group which completely dissolves in water.

R1 and R2 protein powders are mixed according to different ratios, then completely dissolved into 5% solutions using HFIP.

Water resistance testing method: Spider silk film is cut into two pieces of equal weight, each piece placed on the bottom of a container. The control group is placed in a 60 C incubator overnight then weighed (mi). The test group is soaked in deionized water for 30 s, repeated 3 times, then incubated at 60°C overnight before being weighed (m2). The remaining weight of the spider silk film is recorded, and the weight difference between control and test groups calculated in order to determine water resistance. Water resistance is calculated as:

Insoluble matter (%) = mi/mj x 100%

R1 proteins comprise of more /3 -sheet structures, so it is hypothesized that films with higher proportions of R1 would be more water resistant. Water resistance trials show that 100:0 pure R1 films retain their original shape after soaking and retain about 85.1 % of their original mass. 0: 100 pure R2 films completely dissolved in water. Films with higher proportions of R1 exhibit higher levels of water resistance. 75:25 films retained 83.3% of their original mass; 50:50 films retained 81.7% of their original mass; 33:67 films retained 81.2% of their original mass; 25:75 films retained 80.7% of their original mass, and 10:90 films retained 50.3% of their original mass, as illustrated in FIG. 24.

Example 16: Secondary structural ratios of the recombinant spider silk protein

Based on the film water resistance analysis and protein sequence properties, it can be observed that films with higher ratios of R1 are more water resistant. This is because R1 comprises more hydrophobic 3 -sheet structures compared to R2. In order to analyze differences in /5 -sheet structures of films created from different ratios of R1:R2, Fourier-transform infrared spectroscopy (FTIR) (IASCO FT/IR 4100 spectrometer, Tokyo, Japan) is used to analyze the ratios of /3 -sheets and random coil/helix structures in recombinant spider silk films of the present invention.

FIG. 25 illustrates the results of FTIR analysis. The yellow dotted line marks 1,622 cm ¹; a peak at this wavenumber represents the presence of ? -sheets in the material. The blue dotted line marks 1,649 cm ¹; a peak at this wavenumber represents the presence of random coil/helix in the material. The proportion of ? -sheets in each film can be approximated by calculating the ratio of these two peaks. The green line represents pure R1 film (R1:R2 = 100:0), the pink line represents R1:R2 = 75:25, the purple line represents R1:R2 = 50:50, the red line represents R1:R2 = 25:75, and the black line represents pure R2 film (R1:R2 = 0: 100). To approximate the proportion of 3 -sheet and random coil/helix structures within each film, peaks at 1,622 cm'¹ (signifying /? -sheet) and 1,649 cm ¹ (signifying random coil/helix) arc compared. In pure R1 film, the peak at 1,622 cm'¹ is obviously higher than that of 1,649 cm'¹, while the opposite is true for pure R2 film. In 75:25 film, the peak at 1,622 cm ¹ is slightly higher than that of 1,649 cm'¹. In 50:50 film, both peaks are of equal height. In 25:75 film, the peak at 1,649 cm'¹ is slightly higher than that of 1,622 cm ¹. Results indicate that R1 spider silk indeed comprises more - sheet structures than does R2, and that the proportion of /3 -sheet structures within R1 and R2 mixture films is positively associated with the R1:R2 ratio. As such, films with higher proportion of R1 should exhibit higher water resistance and tensile strength, properties related to the presence of -sheet structures.

Example 17: Water absorption of recombinant spider silk proteins

In order to measure water absorption capability of the recombinant spider silk film, films are cut into squares, and soaked in water for 24 hrs to reach full saturation. The surface of each film is then wiped dry, then each piece of film is weighed. The pieces of film are then dried in a drying oven, then weighed again to obtain dry weight of each piece of film. The difference in wet weight and dry weight are calculated as percentage weight gain to find the water absorption of films created from different R1:R2 ratios.

As shown in FIG. 26, 1:0 pure R1 film exhibits lowest water absorption capabilities, while 1:3 film exhibits highest water absorption capabilities. Water absorption could not be measured for pure R2 film, which dissolved in water. 1:0 film absorbed 82.0% its mass, 3: 1 film absorbed 99.6%, 1: 1 film absorbed 112.3%, 1:2 film absorbed 172.7%, and 1:3 film absorbed 337.0%. In addition, it can be observed during the soaking process that R2 composition in films is positively correlated to distortion of film shape during soaking, with 1:3 film exhibiting the most distortion.

Example 18: Tensile testing

Tensile testing is performed on pure R1 and pure R2 films to act as control. FIG. 27A illustrates results of tensile testing on R1 film, with strain (%) on the x-axis and strength (N/mm²) on the y-axis. FIG. 27B illustrations results of tensile testing on R2 film. FIG. 27C compares the strength of R1 film and R2 film. As shown in the graphs of FIG. 27, the average maximum stress of R1 film is 38.5 N/mm², while the maximum stress of R2 film is 30.8 N/mm². It can be observed that R1 film exhibits higher tensile strength than does R2. In terms of ductility, R1 film does not exceed 5%, and the ductility of R2 film is averaged at 10%.

Example 19: Heatability of recombinant spider silk protein

As R2 recombinant protein comprises more amorphous long chain structures, an R2 film ripped by an external force may self-heal. R1 recombinant comprises less amorphous structures, and as such exhibits lesser self-healing capabilities. Films of different R1 :R2 proportions are created for healability tests illustrated in FIG.

28. The original film group is cut into appropriate shape and size, then subjected to tensile tests to analyze tensile strength. The healed film group (also known as healing film group) is cut into appropriate shape and size, slashed, healed, and dried prior to tensile testing. In other words, films of the healed film group are cut in half, the area to be healed is dipped in deionized water, and the dipped portions of the cut films are overlapped for self-healing. Films of the healed film group are placed in a dry cabinet. After tensile testing, the healed films are inspected to ensure that the new rips did not occur in the self-healed area. Only healed films with stock-break failure separations following tensile testing are considered to have been effectively healed. Tensile strength is calculated.

Healability is calculated as:

(Strength of healed film) / (Strength of original film) x 100%

FIG. 29A illustrates the results of tensile testing for both original films and healed films of different R1:R2 ratios. In the original film group, tensile strength of pure R1 film is measured at 38.5 N/mm², 3: 1 film at 35.5 N/mm², 1: 1 film at 33.8 N/mm², 1:2 film at 32.7 N/mm², 1:3 film at 32.1 N/mm², and pure R2 film at 30.8 N/mm². In the healed film group, tensile strength of pure R1 fdm is measured at 14.4 N/mm², 3: 1 film at 24.2 N/mm², 1: 1 film at 28,4 N/mm², 1:2 film at 30.9 N/mm², 1 :3 film at 31.4 N/mm², and pure R2 film at 30:3 N/mm².

FIG. 29B illustrates healability of recombinant spider silk films of different R1:R2 ratios. After tensile testing of healed films, all but the films with stock-break failure are eliminated, and healability is calculated by dividing strength of healed film by strength of original film. As shown in FIG. 29B, pure R1 film exhibits lowest hcalability, recovering only 37.4% tensile strength, while pure R2 film exhibits highest healability at 98.3% tensile strength. The healability of 3: 1 film is 68.2%, 1: 1 film is 84.1%, 1:2 film is 94.5%, and 1:3 film is 97.7%.

Although pure R2 film exhibits highest hcalability, recovering 98.3% tensile strength, its application is limited due to its tendency to completely dissolve in water. However, a film with R1:R2 ratio at 1:3 exhibits nearly as high a healability, and is sufficiently water resistant, retaining its shape and mass in water to a certain degree.

Example 20: Protein film pipe Using recombinant spider silk film of R1 :R2 = 1 :3, which exhibits highest healability and is insoluble in water, a protein film pipe is created by rolling the film and allowing it to self-heal, as shown in FIG. 30. Tests are performed to examine the protein film pipe’s application as a water pipe. A hole is cut into the pipe to simulate damage in the protein film pipe, and a new piece of film is used to patch the damaged section. After patching, water is allowed to flow through the pipe in order to examine the protein film pipe’s water resistance as well as results of patching.

FIG. 31 A-C illustrates a protein film pipe created from one layer of recombinant spider silk film. Thickness of the film is 200 fl m, the inner diameter of the pipe is 4.5 mm, and the flow of water is set to 150 mL/min. As shown in FIG. 31 A, spider silk film is rolled into a protein film pipe, and some water is applied to the overlapping region to facilitate self-healing. Afterwards, the pipe is allowed to dry in a dry cabinet for at least 1 day.

Water flow devices are created as shown in FIG. 3 IB, wherein the protein film pipe is placed in the center, and metal pipes are used to connect the protein film pipe to plastic pipes. A peristaltic pump is used to continuously pump water into the pipes, with water flow set to 150 mL/min. The pipes are observed for 1 hr. Within 30 min, the protein film pipe is observed to contract inwards, although no leakage or damage to the pipe has been observed, and the protein film pipe docs not contract further. This contraction is attributed to the film becoming softer after absorbing water, as well as structural instability.

FIG. 31C demonstrates the flow of water within the pipe system.

Example 21: Protein film pipe (two-layer)

In order to solve the issue of pipe contraction, a protein film pipe is created from recombinant spider silk film twice the thickness of that in Example 20. As shown in FIG. 32, one layer of film is rolled into a cylindrical shape, then a second layer of film is used to cover the first, with some water applied to facilitate healing into pipe-shape as well as healing between the first and second layers. The two-layer protein film pipe is placed in a dry cabinet for at least 1 day.

FIG. 33A-C illustrates the finished protein film pipe (two-layer). The total thickness of the film is 400 /z m, the inner diameter of the pipe is 4.5 mm, and the flow of water is set to 150 mL/min. The pipes are observed for 2 hrs, during which no contraction or obvious shape change is observed, as illustrated in FIG. 33B. FIG. 33C illustrates the flow of water through the protein film pipe.

A hole is cut into the two-layer protein film pipe to simulate possible damage to the pipe. The diameter of the puncture is about 1 mm. When water flow is turned on, leakage is observed from the pipe. Then a piece of new film 100 /z m thick, 3 mm by 3 mm in area is used to patch the hole. Slight pressure is applied for 10 s to ensure that the new film stays in place, then water flow is immediately turned on for 2 hrs to examine results of healing. The process is illustrated in FIG. 34A-C. During the 2 hrs of continuous water flow, no leakage is observed from the protein film pipe, indicating that the protein film pipe can be easily patched within a short frame of time even if damage occurs, and can be put back to use immediately.

It can be appreciated by those skilled in the art that changes could be made to the examples described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims

What is claimed is:

1. A recombinant spider silk protein (rSSP) comprising a repeating REP domain wherein the REP domain comprises one or more repeating units; wherein each of the repeating unit comprises a R1 protein or a R2 protein; wherein the amino acid sequence of the R1 protein is at least 80% similar to SEQ ID NO. 3; and wherein the amino acid sequence of the R2 protein is at least 80% similar to SEQ ID No.

4.

2. The recombinant spider silk protein of claim 1, further comprising a non-repeating N- terminal domain (NTD) and a non-repeating C-terminal domain (CTD) wherein the REP domain is flanked by the NTD and the CTD wherein the amino acid sequence of the NTD is at least 80% similar to SEQ ID NO. 1; wherein the amino acid sequence of the CTD is at least 80% similar to SEQ ID NO. 2;

3. The recombinant spider silk protein of claim 1, wherein the REP domain comprises one or more of the R 1 protein and one or more of the R2 protein at a ratio of numbers of the R1 protein to numbers of the R2 protein of about 3: 1 to about 1:5.

4. The recombinant spider silk protein of claim 1, wherein the REP domain comprises one or more of beta-sheet crystalline structures and one or more of amorphous structures at a ratio of number of the beta-sheet crystalline structures to number of the amorphous structures of about 75:25 to about 55:45.

5. The recombinant spider silk protein of claim 4, wherein the amorphous structures comprises beta-turn, beta-spiral, 3i-helix or a combination thereof.

6. The recombinant spider silk protein of claim 1 , wherein the REP domain comprises about 20 to 60% beta-sheet crystalline structure, about 0% to 60% beta-turn structure and about 0% to 60% 3 i-helix/beta- spiral structure.

7. The recombinant spider silk protein of claim 1, wherein the spider silk protein exhibits self-healing capabilities.

8. The recombinant spider silk protein of claim 1, wherein the spider silk protein is blended with other materials such as graphene, graphite, carbon nanotubes, metal oxides, ceramics and/or other synthetic nanoparticles.

. The recombinant spider silk protein of claim 1 , wherein the REP domain comprises up to 300 repeat units.

10. The recombinant spider silk protein of claim 1, wherein the REP domain comprises at least 60 repeat units.

11. The recombinant spider silk protein of claim 1 , wherein the molecular weight is from about 5 kDa to about 800 kDa.

12. The recombinant spider silk protein of claim 1, wherein the protein is tag-free.

13. The recombinant spider silk protein of claim 1, wherein the toughness of the protein is up to about 250 MJ/m³.

14. The recombinant spider silk protein of claim 1, wherein the recombinant spider silk protein may be purified without the use of affinity or diffusion-based methods such as columns, chromatography, dialysis, and the like, and wherein the resulting recombinant spider silk protein is up to about 80%, 85%, 90%, 95% or 98% pure.

15. The recombinant spider silk protein of claim 2, wherein the amino acid sequence of the NTD is modified at D35K and K60D;

16. The recombinant spider silk protein of claim 1, wherein the purified recombinant spider silk protein is not denatured.

17. A method for purifying the recombinant spider silk protein of claim 2, comprising the steps of: i. Lysing a prokaryotic or eukaryotic system comprising the recombinant spider silk protein using a lysis buffer wherein the prokaryotic or eukaryotic system is capable of expressing the recombinant spider silk protein; ii. Centrifuging resulting lysis solution to obtain cell pellet; iii. Resuspending the cell pellet of step ii in lysis buffer; iv. Sonicating the solution of step iii on ice; v. Centrifuging the solution of step iv to obtain the cell pellet; vi. Resuspending the cell pellet of step v using sodium dodecyl sulfate (SDS) buffer; vii. Sonicating the solution of step vi on ice; viii. Centrifuging the solution of step vii to obtain a supernatant; ix. Centrifuging the supernatant of step viii to obtain the recombinant spider silk protein; and x. Washing the recombinant spider silk protein of step ix using deionized (DI) water or ammonium bicarbonate buffer at least 3 times.

18. The method of claim 17, wherein the prokaryotic or eukaryotic system comprises bacteria, yeast, mammalian cells, plants, insect cells and/or transgenic animal cells capable of expressing the recombinant spider silk protein of claim 1.

19. The method of claim 17, wherein the prokaryotic or eukaryotic system comprises E. coli cells.

20. The method of claim 17, wherein the method of claim 17 is completed in under about 12 hours.

21. The method of claim 17, wherein purity of the recombinant spider silk protein is up to about 85%, 90%, 95%, 98%, 99% or up to 100%.

22. The method of claim 17, wherein the purified spider silk proteins remain stable when stored at about 4°C or lyophilized for long-term storage of more than about 1 month, 2 months, 3 months in non-lyophilized form or for about 6 months, 8 months, 10 months or 1 year in lyophilized form.

23. The method of claim 17, wherein the purified recombinant spider silk protein is not denatured.

24. The method of claim 17, wherein the purified the recombinant spider silk protein is capable of forming micelles.

25. The method of claim 17, wherein the lysis buffer comprises about 0.1 to 0.5 mg/mL lysozyme, about 0.1 to 1.0 wt% Triton X-100, and about 10 to 100 mM ammonium phosphate.

26. The method of claim 17, wherein the SDS buffer comprises about 1 to 20 wt% sodium dodecyl sulfate, about 0.1 to 1.0 wt% Triton X-100 and about 1 to 20 mM Tris-Cl.

27. A recombinant spider silk protein (rSSP) comprising a repeating REP domain wherein the REP domain comprises one or more beta-sheet crystalline structures and one or more amorphous structures at a ratio of numbers of the beta-sheet crystalline structures to numbers of the amorphous structures of about 75:25 to about 55:45.

28. The recombinant spider silk protein of claim 27, wherein the amorphous structures comprises beta-turn, beta-spiral, 3i-helix or a combination thereof. The recombinant spider silk protein of claim 27, wherein the REP domain comprises repeating units wherein each repeating unit comprises either R1 protein or R2 protein; wherein the amino acid sequence of the R1 protein is at least 80% similar to SEQ ID NO. 3; and wherein the amino acid sequence of the R2 protein is at least 80% similar to SEQ ID No.

4.