Directed Evolution of Protein-based Biomaterials Enabled by Advances in High-throughput Mechanical Testing and Protein Purification

Chen, Yusu

doi:https://doi.org/10.21985/n2-8hvv-1d87

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Directed Evolution of Protein-based Biomaterials Enabled by Advances in High-throughput Mechanical Testing and Protein Purification

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Protein-based biomaterials are widely used in biomedical applications and mechanical support because of their novel structural flexibility, biocompatibility and mechanical properties. Protein-based biomaterials outperform traditional synthetic materials in various environments as traditional materials lack the diverse chemical functionalities that proteins offer. Novel bioinspired techniques such as directed evolution offer the most promising path toward engineering adhesive materials because it does not require prior knowledge of the sequence-function relationship. However, the current material characterization techniques do not have the required throughput to use these biological techniques to engineer biomaterials. This thesis focuses on bringing synthetic biology techniques to the materials science community by providing high throughput tools for biomaterial protein production and mechanical testing.The first part of this thesis describes two novel high throughput centrifugal mechanical testing methods for adhesive properties and bulk mechanical properties. Centrifugation can apply a homogenous mechanical force across all the wells of the multiwell plate. Coupled with microparticles that are either attached to the adhesive films or embedded in the gels cast inside wells, the centrifugal detachment force on the particles can be used to measure the specific properties of interest. For the adhesive test, we found that the centrifugation force can successfully differentiate films with various adhesive strengths. Moreover, the adhesive properties measured using the centrifugal test correlated well with the average adhesive forces measured by the standard probe-tack test. We were able to achieve a throughput of 1536 samples per run with our technique, and the throughput could be potentially increased to 6144 samples per run if using a 1536-well plate. For the bulk mechanical test, we found our method can quantitatively measure the yield stress of materials, matching our theoretical prediction. Moreover, we developed a separable glass-bottom well plate method to apply the method to virtually all gel systems. In the end, the high throughput centrifugal bulk mechanical test was shown to have a similar throughput as the adhesion test. Both of these two tests can potentially revolutionize novel materials development as they significantly expedite the material characterization process. The second part of this thesis focuses on the development of a high throughput pure adhesive protein production method that utilizes elastin-like polypeptide (ELP) as the purification tag. ELP has been successfully used to purify various proteins and is attractive for low-cost multiplex purification. To enable purification of the adhesive mussel foot protein-5 using ELP, we explored different ELP-Mfp5 constructs for the best performer. We optimized the solubilization and expression conditions to ensure a good soluble protein yield from bacterial expression. We then screened different precipitants for the optimal purification strategy. We successfully identified ammonium sulfate and ethanol as the precipitants respectively for the first round and the second round of purification. The final yield of the purification is ~30 mg/L culture, which is 5x the amount required for the characterization. In the end, we also explored the self-assembly properties of the virus-like particle MS2. The MS2 particle has been attractive for drug delivery purposes because of its tunable size. To further our understanding of the self-assembly properties, we constructed a saturation mutagenesis library of the MS2 S37Y mutant, which has been shown to form a fibril structure. We developed a flow cytometry-based assay to select for the fibril-forming library members, and we have optimized the FACS parameters and the NGS sample preparation steps. The next step is to screen the library and construct the fitness landscape of the MS2 S37Y mutant.

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