Engineering

Research

It is shown in the research that Ultrabithorax (Ubx) possessed the unique in vitro features, which have given it the advantages of developing versatile protein-based biomaterials. Ubx is biocompatible, biodegradable and non-immunogenetic.1 Those protein properties have highlighted their possible use in biomedicine. Furthermore, Ubx possesses the ability to self-assemble into nanoscale fiber by forming crosslinking dityrosine bonds. It is suggested that dityrosine-containing motifs are the domains contributing to most of its self-assembly.2 Lastly, a study shows that Ubx can be readily functionalized with other proteins and retain its structure stability and mechanical properties afterwards.3

Image

In our lab work, our aim is to develop a novel biomaterial with dityrosine containing motifs of protein Ultrabithorax (Ubx). We expected the product to retain the self-assembly and mechanical properties when engineered with functional protein mRFP, which further demonstrated its potential to incorporate diverse functional proteins for wide applications in various fields in future. Therefore, we conducted 3 engineering cycles.

Cycle 1: Ubx

Design

To obtain insights associated with Ubx protein properties, we created a composite part K4377006. (Figure 1). First, we attached 10x His tag at the N terminal of Ubx in order to purify the protein after expression. The attachment of 10x His tag on N terminal instead of C terminal is for the purpose to further amplify out production of Ubx. Second, we performed codon harmonization to reduce protein incorporation in expressed bacteria. The optimized sequence of codons is expected to demonstrate better expression by our engineered E. coli.

Figure 1.Composite part K4377006. This composite part contains constitutive promoter, RBS, and Ubx protein.

Build

Cloning result

We successfully amplified Ubx plasmid in E. coli DH5α and transformed into E. coli BL21 for expression.

Figure 2.Ubx plasmid extracted from E. coli BL21 digest check result. M - Marker. 1 - Ubx plasmid extracted from E. coli BL21 digest check result, backbone (pSB1A3, 2114bp) / insert (composite part K4377006, 1475bp).

SDS-PAGE result

After expression, we conducted SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to verify whether the protein was produced (result in Figure 3).

Figure 3.Composite part K4377006 SDS-PAGE result. M - Marker. 1 - Control, pSB1A3 plasmid. 2 - Ubx plasmid, target band Ubx 42.56 kDa.

Test

As shown in the SDS-PAGE result, the production was below expectation. We further conducted functional tests, but there was no significant evidence that the protein is able to form dityrosine bonds.

Learn

After discussion, we decided to focus on finding approaches to achieve better production. Thus, we designed engineering Cycle 2.

Cycle 2: Binding Region Prediction

Design

In cycle 2, we developed approaches to narrow down to dityrosine containing domains among Ubx sequence, as dityrosine cross-linkings are evidenced to drive the assembly of proteins and peptides.4 In step 1, we used the ANCHOR algorithm to predict the region among Ubx sequences that demonstrate better binding. On the other hand, a paper suggested that the position of lysine will affect the rate of formation of dityrosine bonds.5 This has further led us to our step 2 - building a model to precisely predict exact formation of dityrosine cross linking among the possible binding sites.

Build

After the analytics of the ANCHOR algorithm in step 1, we further utilize the results and build a selection model to analyze the distance of lysine and tyrosine. The selection model has further specified the binding probability of tyrosine. Based on the step-wise manner of our design, by inputting a baseline value of binding possibility and the sequence to be analyzed, the model will output a line chart based on the list below.

Figure 4.This is the initial output from ANCHOR algorithm, the first row represents the number and the kind of amino acids in the whole sequence. The second row represents the binding probability, the third row represents whether the amino acid has been chosen as the possible binding region, inside the yellow column, is the sequence of Y167 short peptides, which are defined by Figure 5, and the red column shows the peptide Y167 and its probability.

Test

Figure 5.According to Figure 4, our script will help you generate and check the value then form a line graph.Upon the baseline, we can see two regions with tyrosine at the peak, which represent Y167 and Y240.

Learn

According to the result of the ANCHOR algorithm and the model we built, Y167 region and Y240 of the Ubx showed the best probability of forming dityrosine cross linking.6 Thus, we further engineer Y167 and Y240 in our cycle 3.

Cycle 3: Y167 mRFP Y240

Design

With the result of cycle 2, we chose to further design Y167 and Y240 with mRFP in our cycle 3. We first engineered functional protein mRFP (Part:E1010) with Y167 Y240 short peptides in the attempt to verify the self-assembly function. We chose mRFP because it is easy to observe. Second, we attached 10x His tag at the N terminal of Ubx protein in order to purify our protein after expression. The reason we add 10x His tag at N terminal but not C terminal is to amplify the production of Ubx protein. Lastly, we performed codon harmonization to reduce protein incorporation in expressed bacteria. The optimized sequence of codons is expected to demonstrate better expression by our engineered E. coli.

Figure 6.Composite part K4377009. This composite part contains a constitutive promoter, RBS, 10x His tag, mRFP, and two functional domains of Ubx protein.

Build

Cloning result

With the same process of building Composite part K4377006, we successfully built Composite part K4377009.

Figure 7.Y167 mRFP Y240 plasmid extracted from E. coli BL21 digest check result. M - Marker. 1 - Y167 mRFP Y240 plasmid extracted from E. coli BL21 digest check result, backbone (pSB1A3, 2114bp) / insert(composite part K4377009, 1066bp).

SDS-PAGE result

After expression, we conducted SDS-PAGE to verify the protein was produced (result in Figure 8).

Figure 8.Composite part K4377009 SDS-PAGE result. M - Marker. 1 - Control, pSB1A3 plasmid. 2 - mRFP control, E1010 on pSB3K3, target band mRFP 25.4 kDa. 3 - Y167 mRFP Y240 plasmid, target band Y167 mRFP Y240 30.89 kDa.

Test

Solubility of Y167 mRFP Y240

The pellet of Y167 mRFP Y240 plasmid remained red after ultrasonic disruption and centrifuged in PBS buffer. It is assumed that Y167 mRFP Y240 might be insoluble. Therefore, we conducted SDS-PAGE to the amount of Y167 mRFP Y240 in the pellet and supernatant respectively.

Figure 9.Solubility of Y167 mRFP Y240 test. M - Marker. 1 - Control, pSB1A3 plasmid in PBS buffer. 2, 3 - Y167 mRFP Y240 plasmid pellet in PBS buffer, target band Y167 mRFP Y240 30.89 kDa. 4 - Control, pSB1A3 plasmid supernatant in PBS buffer. 5, 6 - Y167 mRFP Y240 plasmid supernatant in PBS buffer, target band Y167 mRFP Y240 30.89 kDa.

The result showed the pellet contains more amount of Y167 mRFP Y240 than supernatant in the PBS buffer. In order to obtain more amount of Y167 mRFP Y240, we ultrasonic disrupted Y167 mRFP Y240 pellet and control (pSB1A3 plasmid pellet) in 8M Urea (high concentration chaotropic agent). After disruption, we centrifuged the sample and separated the supernatant to dialyze into Tris Base buffer and ran SDS-PAGE (result in Figure 10).

Figure 10.Y167 mRFP Y240 in Tris Base buffer. M - Marker; 1 - Control, pSB1A3 plasmid supernatant in Tris Base buffer. 2, 3 - Y167 mRFP Y240 plasmid supernatant in Tris Base bufferTrisbase buffer, target band Y167 mRFP Y240 30.89 kDa.

Also, we measured the mRFP intensity of the supernatant in both PBS and Tris Base buffer (result in Figure 11).

Figure 11.mRFP intensity of the supernatant in both PBS and Tris Base buffer. As Figure 11 shows, mRFP intensity of the supernatant in Tris Base buffer is way higher than the one in PBS buffer, as a result, we conduct the same process for further experiments.

As Figure 11 shows, mRFP intensity of the supernatant in Tris Base buffer is way higher than the one in PBS buffer. The result shows that mRFP (as functional protein) remains its function after it is fused with two short peptide areas. Then, we conducted the same process for further experiments.

Functional Test

To prove that our protein crosslinks with dityrosine bonds. We confirmed the formation of dityrosine bonds from FT/IR and CD measurements of our sample. Later, we explored the application of our biomaterial. Viscosity test results showed that our sample has potential to be bioink. We later 3D printed our sample and acquired the ideal outcome.

Figure 12.Macroscopic testing with different concentration of H2O2 and EDTA-Fe
Figure 13.Dityrosine forming process

We used FT/IR-4700 to analyze additional and reduced bondings before and after processing with EDTA-Fe and H2O2. Data proves crosslinking reaction came from dityrosine bond formation with the obvious decrease in C-H bonds. (result in Figure13.)

Figure 14.FT/IR graph of our protein

We did measurement of viscosity for our protein sample and confirmed its shear thinning behavior, which shows high potential for the future development of bioinks for 3D printing.

Figure 15.FT/IR graph of our protein. (a) Viscosity vs. shear rate. (b)The result of 3D printing.

Learn

Through functional tests, we had successfully achieved our goal of creating the biomaterial that can crosslink and preserve the function of functional protein fusion with them. It is concluded that our biomaterial has the potential to successfully incorporate diverse functional proteins for wide applications in various fields.

Reference

  1. Pavlopoulos, A., & Akam, M. (2011, February 15). Hox gene ultrabithorax regulates distinct sets of target genes at successive stages of drosophila haltere morphogenesis. Proceedings of the National Academy of Sciences of the United States of America.
  2. Alexandra M. Greer, Zhao Huang, Ashley Oriakhi(2009, March 18). The Drosophila Transcription Factor Ultrabithorax Self-Assembles into Protein-Based Biomaterials with Multiple Morphologies Biomacromolecules 2009 10 (4),829-837
  3. Tsai, S.-P., Howell, D.W., Huang, Z., Hsiao, H.-C., Lu, Y., Matthews, K.S., Lou, J. and Bondos, S.E. (2015), The Effect of Protein Fusions on the Production and Mechanical Properties of Protein-Based Materials. Adv. Funct. Mater., 25: 1442-1450.
  4. Fang J, Mehlich A, Koga N, Huang J, Koga R, Gao X, Hu C, Jin C, Rief M, Kast J, Baker D, Li H. Forced protein unfolding leads to highly elastic and tough protein hydrogels. Nat Commun. 2013;4:2974. doi: 10.1038/ncomms3974. PMID: 24352111; PMCID: PMC3983047.
  5. Constancio Gonzalez-Obeso, Fredrik G. Backlund, and David L. Kaplan Biomacromolecules 2022 23 (3), 760-765DOI: 10.1021/acs.biomac.1c01192
  6. Howell DW, Tsai SP, Churion K, Patterson J, Abbey C, Atkinson JT, Porterpan D, You YH, Meissner KE, Bayless KJ, Bondos SE. Identification of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax. Adv Funct Mater. 2015 Oct 7;25(37):5988-5998. doi: 10.1002/adfm.201502852. Epub 2015 Aug 31. PMID: 28725173; PMCID: PMC5513195.