Elsevier

Journal of Biotechnology

Volume 126, Issue 2, 1 November 2006, Pages 186-195
Journal of Biotechnology

Cell-free protein synthesis at high temperatures using the lysate of a hyperthermophile

https://doi.org/10.1016/j.jbiotec.2006.04.010Get rights and content

Abstract

Systems for cell-free protein synthesis available today are usually based on the lysates of either Escherichia coli, wheat germ or rabbit reticulocyte, and protein synthesis reactions using these extracts are performed at moderate temperatures (20–40 °C). We report here the development of a novel system for cell-free protein synthesis that can be operated at high temperatures using a lysate of the hyperthermophilic archaeon, Thermococcus kodakaraensis. With the system, cell-free protein synthesis of ChiAΔ4, a derivative of T. kodakaraensis chitinase (ChiA), was observed within a temperature range of 40–80 °C, with an optimum at 65 °C. Corresponding chitinase activity was also detected in the reaction mixtures after cell-free protein synthesis, indicating that the synthesized ChiAΔ4 folded in a proper tertiary structure. The maximum concentration of active ChiAΔ4 synthesized was determined to be approximately 1.3 μg/mL. A time course experiment indicated that the amount of synthesized ChiAΔ4 saturated within 30 min at 65 °C, and energy depletion was suggested to be the main cause of this saturation. We further developed a system for transcription and translation-coupled protein synthesis at high temperatures using a combination of T. kodakaraensis lysate and thermostable T7 RNA polymerase.

Introduction

The production of recombinant proteins in appropriate host cells is now a routine alternative for studying the function and biophysical properties of a given protein. The variety of host cells available has expanded greatly in recent years, and ranges from the bacterial and archaeal prokaryotic cells to the higher eukaryotic cells. However, recombinant protein production in living cells sometimes shares a common drawback when the target protein is toxic and/or incompatible with host cell growth. This often leads to growth retardation of the host strain, low protein yield, or destabilization of the expression vector (Marston, 1986, Goff and Goldberg, 1987, Chrunyk et al., 1993).

Cell-free protein synthesis is a method to synthesize proteins in vitro by using mRNA and the active translation machinery in the cell lysate (Matthaei and Nirenberg, 1961, Dvorak et al., 1967). One of the advantages of this system is that one can utilize and develop the system focusing only on protein synthesis per se, and therefore, highly toxic proteins can readily be produced with in vitro systems (Henrich et al., 1982). Another major advantage is that these systems, with the properly charged tRNAs, allow the synthesis of proteins containing unnatural amino acids (Noren et al., 1989). Other notable features are the relatively short periods of time required for protein synthesis and the rather simple purification procedure following protein synthesis.

At present, there are three major sources of lysates utilized for cell-free protein synthesis: Escherichia coli (Spirin et al., 1988), rabbit reticulocyte (Hempel et al., 2001) and wheat germ (Endo and Sawasaki, 2003). As these lysates originate from organisms living at moderate temperatures, protein synthesis reactions are performed in a temperature range between 20 and 40 °C. Although these systems can be presumed to be sufficient for producing a majority of mesophilic proteins, there are several reasons for one to explore the possibilities of protein synthesis at higher temperatures. A slight elevation in temperature, to an extent that it does not denature the target protein itself, will lead to more rapid protein synthesis. It has been reported that, by using capped mRNA, the reaction temperature of wheat germ extract could be increased up to 37 °C (from 20 °C), and an increased amount of protein synthesis was observed as a result of high-speed protein synthesis (Tulin et al., 1995). In addition, elevated temperatures can be expected to prevent mRNA secondary structures that otherwise might be inhibitory in the translation reaction (Myers and Gelfand, 1991).

In order to develop an in vitro translation system that functions and exhibits stability at elevated temperatures, the use of (hyper)thermophiles as a source of cell lysate is a practical choice. The in vitro incorporation of [35S] methionine into proteins has previously been reported using the lysate of Sulfolobus solfataricus strain MT4 (Ruggero et al., 1993, Condo et al., 1999), suggesting that the lysates from hyperthermophiles have the potential to be utilized for in vitro translation systems. Besides the stability at moderately high temperatures (∼50 °C), development of this type of system using the lysate of a hyperthermophile would greatly expand the temperature range at which cell-free protein synthesis can be performed. This should also make possible the production of highly thermostable proteins that cannot be properly folded at ambient temperature.

We report here the development of a system for cell-free protein synthesis using a lysate of Thermococcus kodakaraensis. T. kodakaraensis KOD1 is a hyperthermophilic archaeon isolated from a solfatara on Kodakara Island, Kagoshima, Japan (Morikawa et al., 1994, Atomi et al., 2004). The organism can grow between 60 and 100 °C with an optimal growth temperature of 85 °C. The broad temperature range at which this organism grows can be expected to provide an advantage in developing a cell-free system that can function at various extents of elevated temperature. In this study, we have performed an initial examination of various parameters and components that affect the rate and yield of protein synthesis, and with this system we have been able to observe the in vitro production of an active protein at temperatures between 40 and 80 °C.

Section snippets

Chemicals

Sulfur, Tris–acetate, ammonium acetate, polyethyleneglycol 8000 (PEG 8000) and potassium phosphoenolpyruvate were purchased from Wako Pure Chemical Industries (Osaka, Japan). ATP, GTP, CTP and UTP were from Sigma (St. Louis, MO). RNase inhibitor was RNAsecure™ from Ambion (Austin, TX). All the other reagents were obtained from Nacalai Tesque (Kyoto, Japan).

Plasmids and mRNA preparation

The template DNA, pTRC1, used for preparing ChiAΔ4 mRNA, was constructed as follows. The XbaI site of pUC118 was removed with the Blunting

Selection of the target protein

ChiAΔ4, a truncated form of T. kodakaraensis chitinase (Tanaka et al., 1999), was selected as the target protein to be synthesized in the T. kodakaraensis cell-free translation system. Chitinase from T. kodakaraensis (ChiA) contains two catalytic domains (Tanaka et al., 1999, Tanaka et al., 2001). ChiAΔ4 (33.8 kDa) is a ChiA derivative containing only the C-terminal endochitinase domain. As ChiAΔ4 is a highly thermostable enzyme with a half-life of over 3 h at 100 °C (Tanaka et al., 1999), the

Discussion

The present study reports the development of a system for cell-free protein synthesis at high temperatures using T. kodakaraensis S30 extract. Synthesis of ChiAΔ4 was detected by Western blot analysis in a temperature range between 40 and 75 °C (Fig. 3A). ChiAΔ4 could not be detected by Western blot analysis at 80 °C, while chitinase activity at 80 °C was almost the same as that detected at 40 °C (Fig. 3B). The activity observed at 80 °C may be due to degradation products of ChiAΔ4 that still

References (34)

  • D. Ruggero et al.

    In vitro translation of archaeal natural mRNAs at high temperature

    FEMS Microbiol. Lett.

    (1993)
  • T. Tanaka et al.

    Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

    J. Biol. Chem.

    (2001)
  • H. Atomi et al.

    Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1

    Archaea

    (2004)
  • P. Carninci et al.

    Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA

    Proc. Natl. Acad. Sci. U.S.A.

    (1998)
  • I. Condo et al.

    Cis-acting signals controlling translational initiation in the thermophilic archaeon Sulfolobus solfataricus

    Mol. Microbiol.

    (1999)
  • H.F. Dvorak et al.

    Purification and properties of 2 acid phosphatase fractions isolated from osmotic shock fluid of Escherichia coli

    Biochemistry

    (1967)
  • T. Fukui et al.

    Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes

    Genome Res.

    (2005)
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