Elsevier

Biotechnology Advances

Volume 31, Issue 6, 1 November 2013, Pages 797-803
Biotechnology Advances

Research review paper
Cell-free platforms for flexible expression and screening of enzymes

https://doi.org/10.1016/j.biotechadv.2013.04.009Get rights and content

Abstract

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.

Introduction

As demonstrated by recombinant DNA and PCR techniques, in vitro applications of biological systems have tremendously impacted the progress of modern biotechnology. Cell-free protein synthesis recently has been added to the arsenal of biotechnological methods. Without being constrained by cell viability and membrane integrity, cell-free protein synthesis has the advantages of greater speed and flexibility compared with cell-based gene expression approaches. Moreover, the physicochemical environment of cell-free protein synthesis can be controlled, enabling functional analyses of ribosomes and accessory factors under a variety of reaction conditions. Due to its open nature, cell-free protein synthesis is conducive to virtually unlimited reaction formats (Fig. 1), including homogeneous solution-phase reactions, protein synthesis within artificial vesicles (Griffiths and Tawfik, 2000, Griffiths and Tawfik, 2006, He et al., 2008, Hovijitra et al., 2009, Noireaux and Libchaber, 2004, Yu et al., 2001) and solid-phase protein synthesis in gel matrices (Kim et al., 2009, Kwon et al., 2008, Park et al., 2009). Modularization of reaction components in cell-free protein synthesis also enables the expression of surface-tethered DNA, which has been applied to in situ generation of protein arrays (Byun et al., 2012, He et al., 2008, Ramachandran et al., 2004, Stoevesandt, 2012, Tao and Zhu, 2006).

The in vitro utilization of protein biosynthesis machinery is not a completely novel concept, but cell-free synthesis has only recently been accepted as a meaningful tool for protein production (He et al., 2011, Kim et al., 1996, Schwarz et al., 2007, Schwarz et al., 2008). Since its original uses for deciphering genetic codes 50 years ago (Nirenberg and Matthaei, 1961), the applications of cell-free protein synthesis systems have been limited to studies in which the generation of analytical amounts of proteins was sufficient. It has long been considered that protein biosynthesis machinery was not robust in a cell-free context and could not function long enough to synthesize substantial quantities of proteins. However, driven by a recent upsurge in sequence information, cell-free protein synthesis systems that avoid the bottleneck steps inherent to cell-based expression methods have garnered renewed attention and attempts to improve their productivity. Kim and Swartz suggested that the productivity of batch cell-free protein synthesis reactions was limited by lack of ATP owing to the depletion of energy sources (Kim and Swartz, 1999). Although the ATP level could be augmented by supplying energy sources containing high-energy phosphate bonds, the equimolar accumulation of inorganic phosphates would inhibit protein synthesis by sequestering Mg2 + ions, which serve as essential cofactors in many nucleotide-dependent reactions. Accordingly, attempts to increase the productivity of cell-free protein synthesis techniques have focused on the development of ATP regeneration methods that do not accompany stoichiometric accumulations of inorganic phosphates. They solved this issue by incorporating pyruvate oxidase to catalyze the in situ condensation of pyruvate and inorganic phosphate and generate acetylphosphate, which could be used for ATP regeneration. By this method, inorganic phosphate liberated from acetylphosphate is recycled to form another molecule of acetylphosphate, thereby preventing its accumulation. The subsequent discovery that glucose could be metabolized in cell extracts to regenerate ATP through glycolysis and oxidative phosphorylation became an important milestone in the development of highly productive and cost-effective cell-free protein synthesis systems (Calhoun and Swartz, 2007, Jewett and Swartz, 2004, Jewett et al., 2008, Kim and Kim, 2009, Kim et al., 2007). The glucose-based ATP regeneration system was extended to include an in situ supply of glucose from polymeric carbohydrates (maltodextrin or soluble starch), which provides control over the ATP delivery rate (Kim et al., 2011a, Kim et al., 2011b, Wang and Zhang, 2009). These achievements have remarkably improved the productivity and cost of cell-free protein synthesis techniques and have expanded the applicability of cell-free protein synthesis to fields ranging from high-throughput gene expression to large-scale protein production (Carlson et al., 2012, Katzen et al., 2005, Kim et al., 2006a, Kim et al., 2006b, Sherstha et al., 2012, Yang et al., 2012). In particular, cell-free protein synthesis offers an ideal platform for the expression screening of enzymes (Cohen et al., 2001, Hold and Panke, 2009, Mastrobattista et al., 2005, Shimizu et al., 2006), which is gaining more importance with the progress of industrial biotechnology. Although the expression and screening of genetic libraries of enzymes is generally carried out in microbial cells (Hibbert and Dalby, 2005, Ottosson et al., 2001), the use of living cells sets intrinsic limitations in multiple levels. For example, screening protocols should be designed within the boundaries of physiological conditions and the target enzymes and substrates should not interfere with normal metabolism of the cells. In addition, even when the expressed enzymes do not impede cell viability, the substrates for enzyme screening should be chosen from a narrow range of candidates that are transportable across the cell membrane. These limitation associated with cell-based expression screening can be effectively overcome by expressing enzyme libraries in cell-free protein synthesis systems. Not being subject to the constraints of maintaining cell viability and intact cell membranes, cell-free enzyme synthesis offers greater flexibility in designing the expression and screening procedures. This paper also focused on the recent research activities to implement cell-free protein synthesis technology for discovery and engineering of industrial enzymes. Following a brief introduction to the history and update on the present status of cell-free protein synthesis techniques, representative approaches to apply cell-free protein synthesis systems for enzyme expression are discussed in this article.

Section snippets

Direct translation of in vitro-amplified genes

A unique advantage of cell-free protein synthesis is the ability to translate genetic information without the need for growing cells. However, conventional protocols of cell-free protein synthesis require the use of plasmid templates. Therefore, it is still necessary to grow cells for the cloning and amplification of the DNAs, which off-sets the merit of using a cell-free system. The time- and labor-intensive steps required for plasmid preparation could be avoided if the template DNAs are

Cloning-independent expression screening of enzymes

The integration of in vitro gene amplification and cell-free protein synthesis and in situ analysis holds great potential for enzyme screening from the genomic DNA of microbes (Lui et al., 2011, Stapleton and Swartz, 2010, Woodrow et al., 2006, Yabuki et al., 2007). Kwon et al. (2010) successfully adapted the strategy of cloning-independent cell-free expression to discover novel enzymes from the putative genes from various microbial species (Fig. 3). These researchers proposed an integrated

Cell-free expression screening of mutant libraries

The generation and screening of mutant libraries are indispensable to the discovery of novel biological functions and to the generation of tailored proteins. Although expression screening of mutant libraries has traditionally been conducted in microbial cells, the use of living cells poses intrinsic limitations. For instance, screening protocols must be designed within the boundaries of physiological conditions because cell-based screening is dependent upon cell viability. As such, target

Cell-free synthesis of non-proteinous materials

In cell-free synthesis systems based on crude cell extracts, most soluble cytoplasmic enzymes are present in the reaction mixture together with the key ingredients for protein synthesis. Most of these enzymes can be considered undesirable contaminants because they do not contribute to the process of translation and may even redirect the raw materials of translation to non-productive degradation pathways. However, these “contaminating” enzymes also constitute silent metabolic pathways that can

Summary

By bringing biosynthetic machinery under artificial control, cell-free protein synthesis offers wide latitude of flexibility in terms of manipulating the reaction conditions and formats for protein synthesis. With the recent progress in the developments of highly efficient and economic methods for energy supply and extract preparation methods, cell-free protein synthesis is now being accepted as an alternative option to cell-based expression systems. While such issues as glycosylation and other

Acknowlegement

This work was supported by the National Research Foundation of Korea [grant numbers 2011-0031946 and 2011K000841].

References (60)

  • H.C. Kim et al.

    Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source

    Process Biochem

    (2011)
  • Y.C. Kwon et al.

    Synthesis of functional proteins using Escherichia coli extract entrapped in calcium alginate microbeads

    Anal Biochem

    (2008)
  • B.H. Lui et al.

    Discovery of improved EGF agonist using a novel in vitro screening platform

    J Mol Biol

    (2011)
  • E. Mastrobattista et al.

    High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions

    Chem Biol

    (2005)
  • S. Rungpragayphan et al.

    PCR-linked in vitro expression: a novel system for high-throughput construction and screening of protein libraries

    FEBS Lett

    (2003)
  • T. Sawasaki et al.

    A bilayer cell-free protein synthesis system for high-throughput screening of gene products

    FEBS Lett

    (2002)
  • D. Schwarz et al.

    Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies

    Methods

    (2007)
  • K. Sitaraman et al.

    A novel cell-free protein synthesis system

    J Biotechnol

    (2004)
  • O. Stoevesandt

    Protein arraying by cell-free expression and diffusion across a fluid filled gap

    Nat Biotechnol

    (2012)
  • Y. Wang et al.

    Cell-free protein synthesis energized by slowly-metabolized maltodextrin

    BMC Biotechnol

    (2009)
  • W. Yu et al.

    Synthesis of functional protein in liposome

    J Biosci Bioeng

    (2001)
  • Y.H.P. Zhang et al.

    Biofuel production by in vitro synthetic enzymatic pathway biotransformation

    Curr Opin Biotechnol

    (2010)
  • M.E. Boyer et al.

    Cell-free synthesis and maturation of [FeFe] hydrogenases

    Biotechnol Bioeng

    (2008)
  • M. Bujara et al.

    Exploiting cell-free systems: implementation and debugging of a system of biotransformations

    Biotechnol Bioeng

    (2010)
  • M. Bujara et al.

    Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis

    Nat Chem Biol

    (2011)
  • J.Y. Byun et al.

    In-gel expression and in situ immobilization of proteins for generation of three dimensional protein arrays in a hydrogel matrix

    Lab Chip

    (2012)
  • K.A. Calhoun et al.

    Energy systems for ATP regeneration in cell-free protein synthesis reactions

    Methods Mol Biol

    (2007)
  • Y. Endo et al.

    High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system

    J Struct Funct Genomics

    (2004)
  • M. He et al.

    Printing protein arrays from DNA arrays

    Nat Methods

    (2008)
  • E.G. Hibbert et al.

    Directed evolution strategies for improved enzymatic performance

    Microb Cell Fact

    (2005)
  • Cited by (35)

    • The cell-free system: A new apparatus for affordable, sensitive, and portable healthcare

      2021, Biochemical Engineering Journal
      Citation Excerpt :

      By taking advantage of the unique features of the system, the CFS has enabled, in terms of protein synthesis, high synthesis rate and product yield [7]; post-translational modifications including: the creation of disulfide bonds [8–10], glycosylation [11–17], and site-specific phosphorylation [18,19]; the increase in enzyme functionality and protein stability [20] by: optimizing condition for aggregation-prone proteins [21,22], increasing activity of cofactor proteins [23,24], and the production of hard-to-express proteins including mammalian proteins [8,25–27]; the incorporation of unnatural and non-canonical amino acids into synthesized proteins [28–44]; rapid design-build-test models by expressing recombinant proteins directly from PCR-amplified genes, [45–51] and reusable DNA template platforms [52]; the use of creative methods to maximize production of protein/substrate of interest including: specific compartmentalization of protein synthesis [53–57], the ability to control energy and substrate consumption and regeneration or replenishment [58–63], the ability to control temperature for target protein production [64], the creation of resilient DNA nanotubes [65], and a high tolerance for toxic substrates or products [66]; the production of therapeutic proteins including antibodies [67–72], cancer therapeutics [73–75], stroke and neurological disorders [76,77], and antibiotics [78]. In terms of utilizing the system, the CFS has enabled the ability of prototyping to quickly and meticulously study many biological pathways [79–82] and to closely study the synthesis and function of ribosomes [83,84]; rapidly analyze systems by: rapid enzyme and protein screening [5,6,85–96] and the ease to measure output from input manipulation to convert to robust computational modeling [97,98]; the design of complex genetic circuitry networks [55,56,99–104]; on-demand biomanufacturing [105–108]; point-of-care testing [109–122]; and easy production of educational kits [123–126]. The CFS is also a versatile platform, allowing the focus to be either on protein production, metabolic pathways, or cell studies.

    • Cell-free synthetic biology in the new era of enzyme engineering

      2020, Chinese Journal of Chemical Engineering
      Citation Excerpt :

      By decoupling cell growth objectives from engineered enzyme utilization objectives, the cell-free system provides a controlled and open ideal for directing substrates to a single product [17]. Therefore, the cell-free system has advantages in controlling the reaction environment and also provides greater flexibility in the design and expression of engineering enzymes [18]. At present, cell-free enzyme engineering method is mainly used for the synthesis of functional enzymes that are difficult to synthesize, the screening of enzymes, and the production of chemicals.

    View all citing articles on Scopus
    View full text