To address the limitations of current materials for space exploration—specifically their lack of self-assembling, adhesive, and hydrogel-forming capabilities—a fusion protein was engineered by combining hagfish intermediate filament (HIF) proteins with mussel foot proteins (MFPs). Key outcomes are summarized below:
The fusion protein was successfully heterologously produced in E. coli BL21 (DE3) under the regulation of the conventional T7-LacO operon.
The resulting biomaterial forms threads and hydrogels with high water content, strong adhesiveness, and mechanical resilience.
Comprehensive assessments (protein yield, hydrogel formation, adhesive strength, and structural stability) confirmed its potential as a novel, multifunctional material for future space applications.
Construct Architecture: Two primary HIF variants (α and γ, referred to as rHIFα and rHIFγ) were fused with MFP segments (mfp-3b, mfp-5) using a flexible 10-amino-acid linker (GGGGSGGGS). This linker prevents structural interference and preserves the hydrogel-forming ability of HIF proteins. A 10×His tag (for Ni-IMAC purification) and an enterokinase cleavage site (for tag removal if needed) were added to the N-terminus. All constructs were cloned into the commercial pET-28a vector, with expression controlled by the T7 promoter/terminator and LacO operator (Fig. 1, Fig. 2).
Figure 1. Gene circuit design and fusion protein design.
Figure 2. Expression vector for the fusion proteins, showing only rHIFα-mfp3b as an example
Variant List:
Structural Integrity: 3D modeling via AlphaFold revealed that incorporating MFP segments does not compromise the overall structure or functionality of HIF proteins. The fusion proteins maintain structural integrity and retain essential biological functions (Fig. 3).
Figure 3. Protein Structures predicted by AlphaFold
Construct Verification: The designed DNA constructs were synthesized by Suzhou Antatares Biotech (after safety and feasibility screening) and cloned into the pET-28a vector. Sanger sequencing confirmed that the plasmids were correctly assembled, with sequences matching the intended designs (Fig. 4).
Figure 4. Success in constructions verified by Sanger sequencing. To avoid redundancy, only the results for rHIFα-mfp3b is shown. Other constructs are similar.
Heterologous Expression: Engineered plasmids were transformed into E. coli BL21 (DE3), and protein expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). SDS-PAGE analysis demonstrated that all designed constructs were successfully expressed (Fig. 5A).
SDS-PAGE comparison of soluble proteins (supernatant) and insoluble proteins (pellet, likely inclusion bodies) showed:
Figure 5. SDS-Page results of the designed proteins, for rHIFα, rHIFα–mfp3b, rHIFα–mfp5, rHIFγ, and rHIFγ–mfp5. (A) comparison between uninduced bacteria culture (-iPTG) and induced culture (+) (B) comparison between the soluble protein expressed in the supernatant and the inclusion body protein in the pellet.
Optimization Parameters: To maximize protein yield and solubility, three key parameters were tested in 1 L Erlenmeyer flasks (containing 120 mL TB+Kan medium, 100 μg/mL kanamycin):
Optimal Conditions Identified: SDS-PAGE and grayscale analysis of target bands (Fig. 6A–F) confirmed the optimal conditions as:
Figure 6. SDS-Page results of the various fermentation conditions, using rHIFα–mfp3b, with different (A) IPTG induction concentration, (C) temperature of bacteria culturing after induction, and (E) culturing time after induction. (B), (D), and (F) shows the grey scale of the target band in the supernatant (sup) sample for each of the investigated conditions. The optimal fermentation conditions are determined to be 0.5 mM IPTG, 30°C, and 4 hours.
Initial Challenge: Gravity-driven Ni-TED IMAC column purification initially failed (no protein eluted), as the 10×His tag of HIF proteins was enclosed by unstructured N- and C-termini, limiting Ni-His interactions.
Successful Purification with Urea:
A 3D-printed hollow rack was designed to facilitate the installation and adjustment of gravity-driven Ni columns (Fig. 7A).
Figure 7. Purification with 4M Urea (20 mM Tris-HCl, pH=8.0) lead to successful purification for the proteins. (A) Our team 3D printed a hollow rack to conveniently install and adjust gravity driven nickle column running. (B) SDS-Page of the samples during purification of rHIFγ-mfp5: supernatant (sup), and pellet from after the ultrasonication; sample after twice (2) flow-through (FT2), and with each elution (E1 – E6), we collected 1.5mL protein solution.
Quantitative Measurement (Bradford Assay): Protein concentration in eluted samples was measured, and yield was calculated based on culture volume:
Yield Comparison: The yield of soluble fusion proteins is comparable to previous reports (360 mg/L for rHIF proteins extracted from inclusion bodies [12]). Notably, a portion of the protein remained in the column flow-through, indicating room for further purification optimization.
Co-Assembly Validation: A 1:1 volume mixture of 1 mg/mL rHIFγ-mfp5 and 1 mg/mL rHIFα-mfp3b was centrifuged at 12,000×g for 10 min. White precipitates were observed in the mixture, providing direct evidence of hydrogel formation via co-assembly of the two fusion proteins (Fig. 8).
Figure 8. Hydrogel is formed as white precipitates with 1:1 rHIFγ-mfp5 and rHIFα-mfp3b.
Adhesion Across Materials: rHIFγ-mfp5, rHIFα-mfp3b, and rHIFα-mfp5 exhibited adhesive activity between different material pairs, with varying strength (Fig. 9):
Figure 9. rHIFγ-mfp5, rHIFα-mfp3b, and rHIFα-mfp5 all exhibited adhesive power between different pairs of materials with varying strength: (A), (B) plastics – glass (strong) (C) plastics – metal (weak) (D) glass – glass (very strong) (E) glass – metal (strong).
Adhesion Strength Classification:
Native rHIFα and rHIFγ showed no adhesive strength (samples broke immediately when lifted vertically).
Maximum Adhesive Force:
A LEGO-based force measurement rack (equipped with a force sensor) was used to test adhesion between two glass plates (surface area: 625 mm²). At 0.5 mg/mL:
Figure 10. Measurement of maximum adhesive force. (A) the controlled pulling device with a force measurement sensor using high-school physics lab class devices. The proteins were used as glues between two glass plates, and the surface area is 625mm². The two plates were pulled by the LEGO motor very slowly and the force at which the plates were separated is recorded as the max force. (B) rHIFγ-mfp5 and rHIFα-mfp3b at 0.5 mg/mL showed maximum adhesive force near 7000 N/m², close to normal student glues. Their mixture showed a statistically significant increase in the max adhesive force to almost 10,000 N/m². (***: p<0.001, **: p<0.01)
Concentration Dependence: For rHIFα-mfp3b, maximum adhesive force increased with protein concentration in the range of 0–2.0 mg/mL (Fig. 11). No further increase is expected at higher concentrations.
Figure 11. Maximum adhesive force of different concentrations, with rHIFα-mfp3b. The measurement setup is exactly the same as in the above measurement. In the range of 0 – 2.0 mg/ml concentration, we observed increasing max adhesive force when the concentration increases. We do not expect the trend to continue when the concentration is large enough.
Temperature Range Testing: Adhesive strength of rHIFα-mfp3b was tested under extreme temperatures (simulating space environments):
Figure 12. Maximum adhesive force at different temperatures, with rHIFα-mfp3b. The max force quickly drops to non-adhesive when the temperature reached below -20°C. The max force increase to near 20,000 N/m² when heated upto 150°C. The two low temperature tests were achieved by placing the dried glued glass plates into -20 freezer and -72 freezer in our synthetic biology lab. The high temperature tests were achieved by placing the dried glued plates into the drying oven in our lab, and baked at the respective temperatures for 3 hours. 150°C was reported in the literature to be the temperature on the surface of the space suit when astronauts perform space walking missions.
Low-Temperature Insight: Reduced adhesion at low temperatures is hypothesized to result from decreased DOPA oxidation (a key process for MFP adhesion) under cold conditions.
Truncated Variant Design: Based on literature, removing the unstructured N- or C-terminal regions of rHIF proteins may enhance mechanical strength. The following constructs were designed, and successful cloning was confirmed:
Cloning and Sequencing: Gibson Assembly was used to generate truncated plasmids (Fig. 13.1–13.2). Sanger sequencing verified the correctness of the assembled sequences for rHIFα-δN, rHIFα-mfp5-δN, and rHIFγ-δC (Fig. 14).
Figure 13.1 Gibson Assembly strategy to remove the N- and C- terminal regions from the rHIF proteins.
Figure 13.2: Agarose gel electrophoresis image showing the plasmid successfully assembled using the Gibson assembly method.
Figure 14. Sequencing results verified the successful construction of three gene circuits.
Expression Validation: Truncated proteins were expressed under the optimal conditions identified in Cycle 2. SDS-PAGE analysis showed:
Figure 15. SDS-Page results to verify the induced expression of two rHIF proteins in which the N- terminal regions are removed. For the rHIFα-mfp5-δN construct, the anticipated protein size is around 60 kDa, and clearly shown in the correct region in the page bands. For the rHIFα-δN construct, the anticipated protein size is around 45 kDa, and since there is a strong intrinsic band in the negative control, and also in all other expression, the results are inconclusive on the page results.
Purification: Truncated proteins were purified using gravity-driven Ni columns and 20 mM Tris-HCl + 4 M Urea buffer. SDS-PAGE confirmed successful purification (Fig. 16).
Figure 16. SDS-Page shows the purification sample of rHIFα-δN and rHIFα-mfp5-δN. Through nickle column purification by catching the 10×His tag, we can verify that both proteins are expressed successfully.
Adhesion Confirmation: A simple adhesion test verified that rHIFα-mfp5-δN retains adhesive properties (consistent with its mfp5 segment) (Fig. 17). Quantitative force measurements were not completed due to time constraints.
Figure 17. Simple tests verified rHIFα-mfp5-δN has adhesive power as expected from its mfp5 group. Due to time constraints, we will perform max force measurement in the future.