Session: Research Posters

Paper Number: 173314

A Protein Synthesis Platform for Biomanufacturing of Non-Natural Proteins 

All cellular proteins are synthesized by the ribosome, a molecular machine that uses the genetic information stored in messenger RNA (mRNA) as a template to polymerize different amino acids (AAs) into mature proteins. Regular protein synthesis is driven by random collisions of any mRNA with any ribosome in the cell, making the process of mRNA recruitment by the ribosome completely non-specific. The ribosome has evolved to create proteins from only 20 proteinogenic AAs. But it can also be engineered to site-specifically incorporate non-canonical AAs to produce “designer” proteins with significantly enhanced properties such as increased stability, activity, target binding, and so on. While chemical or cell-free synthesis of such proteins is possible, these approaches suffer from poor scalability and environmental sustainability, making biomanufacturing an attractive alternative. However, scalable biomanufacturing of such proteins remains a challenge because engineering the ribosome for gain-of-function often results in the loss of its ability to synthesize ‘normal’ cellular proteins, leading to cell lethality. To solve this problem, we have genetically engineered Escherichia coli cells to produce two separate populations of ribosomes: I) the endogenous ‘wild type’ ribosomes that supports cell viability and II) a “specialized” ribosome to which an mRNA of interest is covalently attached and is used to synthesize only one protein of interest. Using molecular biology techniques such as Northern blot and primer extension, we show that the ribosome-mRNA chimeric design is expressed and assembled into mature ribosomes with an attached mRNA despite its completely artificial design. Moreover, we demonstrate that the attached mRNA is exclusively recognized by the specialized ribosome to which it is attached. To this end, we deactivate the specialized ribosome via two independent methods - antibiotic treatment and inactivating mutations - to show the expected inhibition in the protein of interest’s production. This indicates that the attached mRNA is not a substrate for, and not recognized by, the wild type ribosome. Finally, we test our specialized ribosomes’ ability to exclusively synthesize different proteins of varying lengths and AA compositions to show their versatility. The high specificity and versatility of our specialized ribosome means that while the endogenous ribosomes remain unmodified and support cell viability, the specialized ribosomes can be engineered extensively to enable the incorporation of non-canonical AAs into the protein of interest, or even for the synthesis of programmed non-protein polymers. Therefore, our unique ribosome-mRNA design can serve as a scalable platform for the biomanufacturing of non-natural proteins with entirely novel properties.

Presenting Author: Kasra Alizadeh Department of Pharmaceutical Sciences and Center for Biomolecular Sciences, University of Illinois at Chicago

Presenting Author Biography: PhD Candidate
Department of Pharmaceutical Sciences and Center for Biomolecular Sciences
University of Illinois at Chicago, College of Pharmacy

Authors:

Kasra Alizadeh Department of Pharmaceutical Sciences and Center for Biomolecular Sciences, University of Illinois at Chicago
Dorota Klepacki Department of Pharmaceutical Sciences and Center for Biomolecular Sciences, University of Illinois at Chicago
Nora Vazquez-Laslop Department of Pharmaceutical Sciences and Center for Biomolecular Sciences, University of Illinois at Chicago
Alexander Mankin Department of Pharmaceutical Sciences and Center for Biomolecular Sciences, University of Illinois at Chicago