Christy Catherine

Cell-free platforms for flexible expression and screening of enzymes.

As was witnessed from PCR technology, in vitro applications of biosynthetic machinery can expand the horizon of biotechnology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocess. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables instant creation of protein molecules from diverse sources of genetic information. Enzyme discovery and engineering is the field of particular interest among the possible applications of cell-free protein synthesis since many of the intrinsic limitations associated with traditional cell-based expression screening of enzymes can be effectively addressed. Cell-free synthesis not only offers excellent throughput in the generation of enzymes, it allows facile integration of expression and analysis of enzymes, greatly accelerating the process of enzyme discovery. This review article is thus intended to survey recent progress in cell-free protein synthesis technology focused on its applications in enzyme expression and screening.

Engineering thermal properties of elastin-like polypeptides by incorporation of unnatural amino acids in a cell-free protein synthesis system

Based on the central dogma of protein synthesis, traditional methods for protein engineering require that altering protein structure and function must be accompanied by changing the nucleotide sequence of the genes encoding the protein. However, the preparation of a template gene for each individual protein requires a great deal of time and effort, thereby limiting the throughput and scope of studying engineered proteins. In this study, we describe translation-level engineering of proteins using cell-free protein synthesis. Taking advantage of the promiscuity of aminoacyl tRNA synthetases in accepting structurally similar amino acid analogues, unnatural amino acids were introduced into elastin-like polypeptides in place of the corresponding cognate amino acids. Through the incorporation of various analogues and starting from the same gene, the phase transition temperatures of elastin-like polypeptides became tunable. Our results demonstrate the usefulness of cell-free protein synthesis for protein engineering using unnatural amino acids without the need for cloning.

Translational Incorporation of Multiple Unnatural Amino Acids in a Cell-Free Protein Synthesis System

Nature uses 20 canonical amino acids as the standard building blocks of proteins; however, the incorporation of unnatural amino acids (Uaas) can endow polypeptide sequences with new structural and functional features. Although aminoacyl-tRNA synthetases (aaRSs) can accept an array of Uaas in place of their natural counterparts, Uaas generally are charged to tRNAs with substantially lower efficiencies. This particularly makes it difficult to incorporate multiple Uaas into a protein sequence. In this study, we discuss the use of a cell-free protein synthesis system as a versatile platform for the efficient incorporation of multiple Uaas into proteins. Taking advantage of the open nature of cell-free protein synthesis that allows flexible manipulation of its ingredients, we explored the application of Uaas in 10 mM range of concentrations to kinetically overcome the low affinity of aaRSs towards unnatural amino acids. Supplementation of recombinant aaRSs was also investigated to further increase the Uaa-tRNA pools. As a result, under the modified reaction conditions, as many as five different Uaas could be incorporated into a single protein without compromising the yield of protein synthesis.

Cell‐free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite‐related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell‐free protein synthesis system for large‐scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high‐throughput expression and identification of individual genes comprising the library, a cell‐free synthesis reaction was designed such that both the template DNA and the expressed proteins were co‐immobilized on the same microbeads, leading to microbead‐based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins.

Enhanced Production of Soluble Recombinant Proteins With an In Situ-Removable Fusion Partner in a Cell-Free Synthesis System

High-yield production of soluble protein is a common concern in diverse fields of biotechnology. In this study, a strategy of using an engineered nucleotide sequence of ubiquitin for enhancing the production of soluble proteins in a cell-free synthesis system is presented. When examined for a series of proteins that otherwise were poorly expressed, N-terminal fusion with ubiquitin significantly increased both the expression levels and solubility of the translational products. The effect of ubiquitin fusion was also markedly augmented by engineering the nucleotide sequence of ubiquitin, leading to several fold enhancements in soluble production of target proteins. Recombinant proteins were produced with their native amino acid sequences through in situ removal of ubiquitin during cell-free synthesis reactions in the presence of a deubiquitinase. The presented strategy could be employed as a facile route to prepare soluble proteins required for various applications.

Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.