Undergraduate Researcher University of Washington Seattle, Washington, United States
Introduction: Currently, there is a reliance on antibodies for a wide range of diagnostic and therapeutic applications due to their high specificity and affinity for target molecules. However, the production of antibodies is expensive and complex, often requiring significant resources and time. A de novo protein design computational pipeline has been established for the design of minibinders. However, these designs are limited in computational efficiency and specificity to their target.
Materials and
Methods: This research aims to develop a novel method of binder engineering using phage-like particles (PLPs) capable of peptide display for biopanning. PLPs are artificial bacteriophage MS2 particles with a synthetic protein capsid and encapsulated mRNA. Using a computational pipeline to design an initial protein scaffold by RFdiffusion, ProteinMPNN and AlphaFold, varying complementary determining regions can be inserted into the scaffold forming variants of binder designs, which can be displayed on the PLP surface. These PLPs, each displaying a variant of the designed binder, form a synthetic phage display library used for biopanning, an affinity enrichment method capable of selecting for a protein binding to a chosen target from a library through successive rounds of selection and washing.
Results, Conclusions, and Discussions: Preliminary experiments have shown the capability to produce phage-like particles capable of high-density display of proteins. Verification of the successful purification of PLP was conducted with SDS-PAGE, where two distinct bands corresponding with maturase and single chain coat protein (the two constituents of the PLP protein capsid) were observed at the expected ratio in yields. Further characterisation of the PLP involved dynamic light scattering, where a size distribution plot by number showed a peak at 30 nm, indicating purified particles are of the correct expected size. Finally, real time PCR was performed to verify that there is mRNA encapsulated within the purified PLPs. In the sample with PLP, the PLP is lysed during the protocol to release RNA, and the RNA is converted to DNA which is then amplified during PCR. Results showed DNA amplification in the sample with PLP, and no DNA amplification in sample with no PLP. Characterisation results all support that the PLPs can be purified at high yields with minimal contamination, and are functional in that they are able to encapsulate DNA. This primes the process of binder display and has good promise for a successful biopanning cycle. Next steps would involve using this construct to display binder designs at high density, and verify the encapsulation of mRNA and identity of the mRNA within the PLP.
Traditional phage-display libraries face limitations due to constraints on peptide size and phage viability. In contrast, phage-like particles offer greater flexibility for displaying larger proteins without constraints of phage viability. The use of a computationally designed protein scaffold with varying complementary determining regions enhances the specificity and efficiency of the binders.
The novel method of using phage-like particles for binder engineering shows promise in overcoming the limitations of traditional phage-display libraries. The ability to display larger proteins and the successful preliminary results in high-density display and purification suggest a successful biopanning cycle. This could result in the production of highly specific binders, offering a more cost-effective alternative to antibodies for diagnostic and therapeutic applications.
