Genome-wide functional genomic analysis for physiological investigation and improvement of cell-free protein synthesis [electronic resource]

We set out to develop and apply a high-throughput cell-free protein synthesis (CFPS) platform that provides functional genomics information for a wide variety of open reading frames (ORFs). We then used this information to improve CFPS yields by 4- to 5-fold, depending on the protein product. With the increasing number of completed genome sequences and ongoing sequencing projects, the post-genomic era has ushered in the promise of complete understanding of biological systems. For such a task, the most important set of information is inarguably the knowledge of the function of each gene product. To lead this endeavor of discerning the properties and functions of the entirety of an organism's genes and gene products, the field of functional genomics has emerged. Functional genomics focuses on dynamic cellular aspects, such as gene transcription, translation, and protein-protein interactions, in attempts to understand the relationship between an organism's genome and its phenotype. Thus, the ultimate goal of such studies is to provide a more complete picture of how biological function arises from the hereditary information of a living system. However, despite this clear interest in analyzing the expression and function of gene products, the development of techniques to address the high-throughput needs of functional genomics has been challenging, given the large diversity of protein functions and physiochemical properties, such as molecular weight and hydrophobicity, as well as the varying expression levels of proteins within a cell. In light of these challenges, we developed a sequential CFPS platform, which is capable of characterizing a variety of diverse proteins in the context of the dynamic metabolic networks that exist in vivo. The first round of expression is directed by PCR-generated expression templates (ETs) and creates an array of cell extracts that are individually enriched with a single target gene product. This round of CFPS is terminated by the selective degradation of the linear DNA templates, and a subsequent round of protein expression is initiated by the addition of a plasmid ET for a reporter protein. The array is then screened to identify gene products that enhanced or inhibited the expression and folding of the reporter. With such a method, we expect that the observations will expand our knowledge of both cell-free and in vivo metabolism, as well as identify key factors and reactions that could potentially lead to improved in vitro transcription, translation and protein folding. CFPS systems offer attractive alternatives to conventional fermentation processes used for protein production. Although improvements in CFPS energetics and reaction conditions have greatly enhanced in vitro protein synthesis, we believe that there are still other issues limiting the productivity of the technology. For this reason, identifying targets that could further improve CFPS is desirable. To validate the developed sequential CFPS protocol, we conducted a genome-wide survey of the well-studied bacterium Escherichia coli (E. coli) to identify soluble gene products that influence the in vitro metabolism. With this method, we identified 139 gene products (79 positive and 60 negative effectors) that influenced the cell-free transcription, translation, and protein folding of our three reporter proteins, as well as the energy metabolism and RNA and protein stability in the CFPS system. Encouragingly, most of the observed effects were consistent with the accepted in vivo metabolic functions of the gene products. However, many were not and required subsequent assays and in-depth literature searches to suggest hypotheses for the in vitro activities of the identified gene products. In many cases, the observations illuminated principles and influences that are unknown, lesser known, or secondary functions that were not expected to influence the CFPS performance. The information from the genome-wide survey was then used to guide modifications of the CFPS system to improve the productivity and duration of in vitro protein synthesis, as well as the efficiency of protein folding. First, fifteen positive effectors were produced and supplemented into the expression reactions in various combinations of the effectors in order to identify cooperative interactions that further enhance system performance. Next, we constructed and evaluated four mutant E. coli strains with chromosomal deletions in non-essential genes that encode negative effectors identified by the genomic survey. We also re-optimized the small molecule metabolite environment in the CFPS reactions. Thus, in the improved in vitro expression system, energy generation, translation initiation and elongation, and protein folding were enhanced; the reaction pH was stabilized; the supplies of specific molecular substrates that are essential for protein synthesis were replenished; and mRNA transcripts were stabilized. With this new system, the total, soluble, and active yields of the several diverse proteins were enhanced by 300 to 400%. The functional genomic analysis of E. coli has greatly broadened our understanding of the biology of the organism. And with the use of species-independent translational leaders that can facilitate cell-free expression (Mureev et al., 2009), our sequential CFPS platform can be used for similar genome-wide surveys of most organisms. In this way, the vast wealth of information available in the sequenced genomes will be utilized, and our knowledge of these biological systems will be significantly improved. Furthermore, the forward, or targeted, metabolic engineering strategy that was used to enhance our CFPS system can be applied to the development and/or improvement of most organism-based in vitro protein expression platforms. These targeted metabolic changes will lead to more rapid and more significant enhancements than traditional improvement strategies, as well as bring us closer to a complete understanding of the biological systems.

Keywords: Chemical Engineering
Advisor: Swartz James R. primary advisor. Thesis advisor
Covert Markus advisor. Thesis advisor
Spormann Alfred M. advisor. Thesis advisor