Part:BBa_K5427001
Keratin31
Background Information
Keratin31 (Krt31) is a well-characterized protein within the keratin family, recognized for its structural role in hair fibers and other keratinized tissues. Its molecular properties and biological functions are well understood, making it a favored target for research and industrial applications. In our project, we chose to clone Keratin31 in E. coli to provide a pure substrate for the characterization of the KerDZ enzyme. Among the various keratin isoforms, we selected Keratin31 for its well-established cloning strategy. Basit et al. (2018) successfully standardized the cloning protocol for Keratin 31 into E. coli DH5α strain, employing a double restriction digestion method—an efficient and reliable genetic engineering approach. This well-documented cloning and expression strategy ensures reproducibility and consistency, making Keratin31 an optimal choice for our future enzymatic experiments.
Design Considerations
To optimize the expression of Keratin31 in E. coli, we applied several strategies. Codon optimization was performed to enhance translational efficiency in the host. Repetitive sequences were manually removed to prevent issues with genetic instability. We ensured that the GC content was within the optimal range for E. coli expression (37%), and illegal restriction sites were removed to comply with Biobrick standards.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Part Assembly
To facilitate the in-house production of pure keratins for future enzyme activity and kinetic studies on KerDZ-induced hydrolysis, we developed a synthetic Keratin31 construct. The Keratin31 gene block, ordered from Integrated DNA Technologies (IDT), includes a tandem polyhistidine (His) tag at the C-terminus of the keratin31 gene, separated by a four-amino-acid spacer. We PCR-amplified the His-tag_keratin31 gene block using forward and reverse keratin-specific pET22b+ primers (Designation #BBa_K5427018 and #BBa_K5427019, respectively) to introduce a Gibson overlapping tail sequence. Concurrently, the vector plasmid pET22b+_T7 was PCR-amplified with forward and reverse pET22b+Keratin primers (#BBa_K5427016 and #BBa_K5427017, respectively) for the same purpose. The PCR-modified vector plasmid pET22b+_T7 and the His-tag_keratin31 gene block were subsequently ligated using NEBuilder HiFi DNA Assembly Master Mix (Lot #10238675) following the standard published protocol.
Characterization of Keratin31
In the last phase of our project we cloned keratin into a pET-22b(+) vector for the production of a pure substrate. This was necessary to our project as it allowed us to quantify macheilies mendes parameters such as kcat, km, and to validate metabolic parameters produced by the dry labs metabolic flux predictions. We also wanted to test the efficiency of our keratinolytic enzyme, and aimed to express keratin in our system (Keratin31).
Figure 1 | Growth curves of DH5ɑ, BL21, and Rosetta-gami strains carrying pET22-b(+)_T7_Krt31 grown in LB Ampicillin. Empty cell controls for each strain were grown in LB. Cultures were grown at 37oC for 10 hours. The Optical Density (OD) at 600nm of each culture was measured every two hours.
Protein Modeling
Keratin31 from sheep (Ovis aries) was selected as the focus of our project as wool is primarily derived from sheep. Keratin was modeled in AlphaFold2 to visualize the structure and to produce a model to use in protein-protein interactions with a keratinase. Overall, the model displayed high confidence. Regions of low confidence represent regions involved in intermolecular interactions within a native wool fiber (Dowling et al., 1986). The Ramachandran plot also implies that the areas of low confidence do not have acceptable phi and psi angles. This was expected due to the nature of interactions of these regions.
Figure 1 | Keratin31 predicted local distance difference test (pLDDT) plot generated by AlphaFold2. Regions of low pLDDT represent hydrophobic regions involved in intermolecular interactions (Dowling et al., 1986).
Figure 2 | Keratin31 multiple sequence alignment (MSA) sequence coverage plot generated by AlphaFold2.
Figure 3 | Keratin31 model generated by AlphaFold2 and visualized with Jmol. GIF was generated by FirstGlance.
Figure 4 | Keratin31 Ramachandran plot generated by SWISS-MODEL.
KerDZ and Keratin Interaction
An HDOCK model was done for KerDZ and keratin. The results also suggested that there are interactions that can occur between KerDZ and keratin. The docking scores are around -200, suggesting the interaction is likely. Furthermore, a confidence above 0.7 suggests a likely interaction (Yan et al., 2020). The ligand rmsd is not a measure of the docking accuracy and is only used to show the difference between the 3D structure of the model and the input structures. This models only one of the many interactions with natural wool proteins. Modeling other interactions of natural keratin proteins with KerDZ may be done.
Figure 5 | KerDZ and keratin 31 interaction generated by HDOCK. Image was generated with SWISS-MODEL.
Table 1 | HDOCK output containing docking score, confidence score, and the ligand rmsd of the top 10 protein-protein interactions for KerDZ and keratin 31.
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