Thomas Burton, Siarra Freuler, Eryn Hanson, & Caleb Thomson w/ Dr Ron Sims, Dr Justin Jones & Dr Randy Lewis
Novel Bio-Adhesives FromRecombinant Hagfish Proteins 𝛂𝛂 & 𝛄𝛄
Abstract Testing Methods and Outputs Analysis of Results
Protein Purification
Formulation and Application
Additional Testing and Statistical Analysis
Future Work
Acknowledgements
Figure 10. Output from Tytron 250 tension LAP testing of alpha-gamma 15% adhesive formulation (purple), alpha 15% (red), and gamma 15% (blue),on
polyester-polyester substrate.
The aim of this project is to create a biologically-based adhesive that is“green”, cost effective, and mechanically viable. Outcomes, in line with theaims, will be to produce spray-on and hydrogel adhesives that areenvironmentally friendly, less expensive than current available bio-adhesives,and at equal or better strength than currently available bio-/traditionaladhesives in industrial applications. This project will be significant in that it isan entirely novel concept involving recombinant hagfish proteins and a novelapplication of them that can address current issues in industrial waste andcost of bio-adhesives. The result of this product will be environmentally clean,economically viable, scientifically innovative, and industrially effective.
The project proved the feasibility of the technology. A number of the attemptsand designs were completely unviable—steel-steel dopes did not survive totesting— and more work must be done to broaden both the application of theproduct, as well as to increase the reliability of the adhesives produced..
• Defining a solvent that is less toxic to be used in mass production and useof the adhesive. The new solvent would enable the adhesive to be appliedin a less well-ventilated area, and lower the protein degradation rate
• Searching for more potential application substrates for recombinant hagfishprotein adhesives, as more substrates would increase the market.
• Additional applications of the hagfish protein will also be explored to findother potential products outside of the pure adhesives realm.
• Commercial production of hagfish protein-based adhesives for industrialuse, work towards a product that could be sold through upscale andperfection of production, isolation and purification of the proteins fromrecombinant sources.
The preparation of the refolded α and γproteins involved successive filtrations using decreasing concentrations of urea and a 50mM ammonium bicarbonate solution before freezing and lyophilization.• Dissolved unfolded proteins were
heated on a hot plate until clear (Figure 1, left)
• Transfer solution to automated apparatus (Figure 2, right)
The primary test if this study was the LAP tensile test performed on a Tytron 250. The outputs for such tests include extension (change inlength of the test materials, in this case in millimeters), force in newtons, and time is seconds. The calculated values also output includeengineering stress (in pascal), engineering strain (% deformation). These values were collected for all adhesives samples on both polyesterand steel substrates, and compared statistically, as will be discussed later. The yield stress, ultimate stress, and break energy of each testwere also calculated, and the average of each was taken and is displayed in the following table (Table 1, below).
020000400006000080000
100000120000140000160000180000200000
0.00E+00 1.00E-02 2.00E-02 3.00E-02 4.00E-02 5.00E-02 6.00E-02
Stre
ss (P
a)
Strain (%)
In addition to LAP testing, each sample was analyzed for absorbance with a UV-Vis Spectrometer. To give accurate, viable results, samples were diluted 500/1,250/1, 125/1, and 100/in in additional 90% formic acid. The results of these testsare shown below. Additionally, a SDS-Page analysis of the protein degradationover time in the formic acid has been attempted, unsuccessfully to date.
Figure 8. Absorbance v. mass concentration graph. The Gorilla Glue (green)absorbance value is at undiluted concentration. Note that 2 of the syntheticdopes—gamma in blue and mixed in purple—exceed the absorbance value ofgorilla glue above 8% mass concentration graph. Not shown: Elmer’s Glue, whichat undiluted has an approximate absorbance value of 1.45 au, more than tripleany other recorded value.
0
0.1
0.2
0.3
0.4
0.5
5 10 15 20
Abso
rban
ce (A
U)
Concentration (%)
Fig 9. Various dope in cuvettes prepared for UV-VisSpectroscopy measurements. Note varying color and clarityof the adhesives. Viscosity differences were equally stark.The solutions shown were initially tested both before andafter curing, but did not yield viable results due to extremeoptical density. They were later diluted as described above.
• No steel-steel material results shown, as the protein adhesives weredeemed unviable for steel adhesion.
• ANOVA tests on all dopes for each individual metric—elastic modulus,yield stress, ultimate stress, and break energy—each give a statisticallysignificant results (p < .01).
0
1
2
3
4
5
6
7
AG 15% AG 20% AG 25% Alpha15%
Alpha20%
Alpha25%
Elmers Gamma15%
Gamma20%
Gamma25%
Gorilla MaterialControl
Elas
tic M
odul
us (M
Pa)
Dope Solution
Thanks to Dr Justin Jones and Dr Randy Lewis for overseeing the project. Thanks to Dr Ronald Sims for providing support to this and many other teams in the
senior design process this year. Special thanks to the USU Spider Silk Laboratory and U.S. Navy for funding the
research, and to the USU Biological Engineering Department for the support, and mentorship. Further
questions can be directed to Thomas Burton at [email protected], the USU Biological Engineering Department, or the USU Spider Silk
Laboratory. For more about the Spider Silk Lab, scan:
Mechanical Properties
Ease of Application Versitility Clarity Total
Weighting Factor 0.5 0.2 0.2 0.1 1
Gorilla Glue 5 5 5 4 4.9
Elmer's Glue 2 4 4 1 2.7
𝛼𝛼-Pure Adhesive 4 3 3 5 3.7
𝛾𝛾-Pure Adhesive 3 3 3 2 2.9
𝛼𝛼𝛾𝛾-Mixed Adhesive 1 3 3 3 2
• Added alternating equal volumes of urea and DI water in 45 minute cycles• Added 50mM ammonium bicarbonate and let run 45 minutes; rinsed with
DI water until conductivity was below 100uS/cm Sample was frozen before lyophilization
• Refolded protein samples experienced __% reduction in mass through polishing step, increasing purity.
0
20
40
60
80
100
120
140
160
180
200
AG 15% AG 20% AG 25% Alpha 15%Alpha 20%Alpha 25% Elmers Gamma15%
Gamma20%
Gamma20%
Gorilla MaterialControl
Yiel
d St
ress
(kPa
)
Dope Solution
0
50
100
150
200
250
300
350
AG 15% AG 20% AG 25% Alpha15%
Alpha20%
Alpha25%
Elmers Gamma15%
Gamma20%
Gamma20%
Gorilla MaterialControl
Ulti
mat
e St
ress
(kPa
)
Dope Solution
ADHESIVE: ELASTIC MODULUS (MPA)
YIELD STRESS (KPA)
ULT STRESS (KPA)
BREAK ENERGY (J)
N
AG 15 2.67 40.67 68.01 866.68 3AG 20 5.67 66.67 77.55 942.43 3AG 25 3.33 25.83 38.14 736.67 3ALPHA 15 6.33 102.17 164.29 3878.16 3ALPHA 20 5.00 77.33 124.04 4426.33 3ALPHA 25 3.67 97.00 110.25 11062.00 3GAMMA 15 5.33 97.00 130.08 2919.00 3GAMMA 20 4.33 60.67 76.58 1138.33 3GAMMA 25 2.33 40.67 64.64 1100.33 3ELMERS GKUE 3.67 61.67 72.76 1082.33 3
GORILLA GLUE 5.33 185.67 225.70 35575.33 3
MATERIAL CONTROL
6.67 178.67 303.92 114153.00 3
90% Formic Acid and refolded alpha and/or
gamma hagfish protein were mixed from 5% w/v up to 25% w/v in
5% increments
Steel shim and polyester film
substrates were chosen for
testing adhesive ability
Protein mixture was added between two substrate strips and heated for 48 hours at 40ºC (Figure 3)
Substrate strips were cut into 1” by 5” strips. 50 microliters of each mixture was added between strips in a 1 in2 area. After heating, the strips were prepared for tensile testing by gluing small wood blocks on each end
of the strips, 10 cm apart. This allowed the testing apparatus to grip the wooden block, reducing slippage measurement error. Wooden blocks were affixed to the
strips using large amounts of gorilla glue, carefully place so as to not run onto the testing area, and clamped in place for a minimum of 24 hours to ensure adequate
adhesion (Figure 4, right).
The successful protein-formic acid mixtures were chosen: 15%, 20%, and 25% (The
mixtures were considered successful if they adhered after heating and held together with
slight tension to the strip).Substrate strips were made with all steel, all polyester, and half steel-half polyester, each common materials used in industry, as well
as underserved by current bio- and biodegradable adhesives. . The polyester
came from a roll off 100% polyester, approximate 1/64th inches think, and the steel
from 1/64th inch think steel shim.
(Right) Figure 3. Oven used for curing adhesives. Trial and error testing lead to a 48
hour curing time for all strips.
Figure 5. Elastic moduli in megapascals comparison for all dopes. Note the high error bars. Compare values to those in Table 1.
Figure 6. Yield stress in kilopascals comparison for all dopes. Note that the Gorilla Glue yield stress exceeded the material control.
Figure 7. Ultimate stress in kilopascals comparison for all dopes. Note that these are the most consistent values of any test, shown
a clear “winner” among the bio-adhesive solutions.
Table 1. Numerical values for all test results Note: while all values are representative of averages, no other statistical analysis is
shown. Standard deviations are represented in respective figures.
