Elsevier

New Biotechnology

Volume 40, Part B, 25 January 2018, Pages 245-260
New Biotechnology

Full length Article
The E. coli S30 lysate proteome: A prototype for cell-free protein production

https://doi.org/10.1016/j.nbt.2017.09.005Get rights and content

Highlights

  • Benchmarking the E. coli S30 lysate core proteome.

  • Classification of proteome subsets relevant for cell-free expression.

  • Tuning S30 cell-free lysate production for improved protein quality.

  • Quantitative proteome analysis of SOS response induced S30 lysates.

Abstract

Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a “black box”. Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures.

This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.

Introduction

During the last two decades, cell-free expression has become a standard platform complementing well established in vivo protein production systems. The inherently open nature of cell-free protein synthesis offers manifold options to enable efficient production of soluble as well as membrane proteins [1], [2], [3], [4], [5]. Due to the lack of boundaries like a cell-wall or cell-membranes, customized artificial expression environments by adding ligands, co-factors or hydrophobic compounds [6], [7], [8], [9], [10] can easily be generated. Difficult proteins having problems in solubility, folding or assembly are preferred targets for cell-free expression. Combinatorial labelling of proteins [11], [12], site-specific insertion of non-natural amino acids [13], [14] and the production of toxins or membrane proteins in artificial environments [15], [16] are further frequent applications. Nevertheless, significant limitations either for production or for rapid product quality analysis still exist due to background activities present in the cell-free reaction lysates.

Very efficient and most frequently used for cell-free expression are lysates prepared from E. coli cells. The lysate preparation has been optimized for high translation efficiency and includes harvesting at logarithmic growth phase and fractionation by different centrifugal forces. Very frequent is centrifugation at 30,000g resulting in the S30 lysate [17], [18], [19], [20]. Furthermore, extensive dialysis and other processing steps result in the precipitation of less stable lysate proteins. The final proteome composition of the S30 lysate as the main system component is thus unknown. However, such knowledge would be valuable for refined processes such as eliminating or suppressing critical enzymes for specific assay development directly in the reaction lysates, implementing sets of identified enzymes for directed product modification or complementing truncated pathways for supporting product formation or quality. In order to address some of these problems, the PURE cell-free expression system has previously been developed by reconstituting the E. coli translation machinery from individually purified components [21]. However, the low expression efficiency indicates that protein production is significantly supported by complex interactions of various lysate components. Proteomic studies of more complex but highly efficient lysates such as S30 are therefore necessary to enable preparative scale product formation in defined environments, to open up new possibilities for tuning reaction conditions and finally for the improvement of product quality [22]. This study presents the first proteome analysis of the highly productive S30 lysate based on the E. coli K12-derivative A19, a primary source for cell-free lysate preparation [20].

The analyzed S30 lysates were prepared by optimized protocols that have already resulted in the production of numerous proteins for structural and functional characterization [4], [18], [22]. However, we further paid attention to cultivation conditions that lead to dynamic proteome changes of E. coli cells in adaption to e.g. stress or starvation [23], [24]. Exposure of E. coli A19 cells to a temperature shift and ethanol induces the SOS response resulting in an increased production of folding chaperones and other potentially beneficial compounds [25]. We have exemplified such an S30 lysate tuning and present a quantitative proteome analysis of S30-S lysates derived from E. coli cells grown under SOS response inducing conditions [26], [27]. The modified S30-S lysate proteomes were characterized according to (i) regulated proteome composition, (ii) protein production efficiency using a standard reporter protein and (iii) folding efficiency of various difficult-to-express model proteins.

Two standard proteomics approaches were implemented to address different underlying questions. First, our main purpose was the qualitative proteome analysis of the standard S30 lysate by focusing on the most sensitive identification strategy to provide an S30 lysate blueprint which was as complete as possible. The result was the identification of 1074 proteins containing a core of 821 proteins repeatedly identified in different S30 lysate preparations. The averaged values of the so-called exponentially modified protein abundance index (emPAI) were taken to provide an approximate and relative abundance estimation of each protein in the lysate, based on the number of sequenced peptides per protein (protein coverage). Secondly, a quantitative proteomics analysis was performed by focusing on the identification of up- and down-regulated proteins in standard S30 lysates relative to S30 lysates derived from E. coli cells after inducing an SOS response (S30-S). The comparison of two distinct S30 lysate samples allows the use of a so-called isotope-coded protein labelling (ICPL) strategy, resulting in a defined mass difference of peptides of the same protein derived from either the S30 or S30-S lysate. As these peptides are analyzed simultaneously, the measured intensity is used to quantify proteins from different samples relative to each other. The ICPL strategy is therefore suitable to identify regulated proteins, while the emPAI value compares different proteins in the same sample based on protein coverage and is thus used to estimate the overall protein diversity and abundance [28], [29]. The general validity of the emPAI value as an approximate estimation of protein abundance is accepted and routinely used [30], [31], [32]. Nevertheless, protein characteristics such as ionizability or hydrophobicity can influence this value in individual cases and this should be considered upon interpretation. The ICPL strategy compares only identical peptides or proteins of different samples simultaneously and is therefore not protein dependent. However, and in contrast to the emPAI values, ICPL provides no quantitative information about different proteins within a lysate sample and is therefore not suitable for an overview study of total protein composition.

This study provides, for the first time, a qualitative analysis of the cell-free S30 lysate proteome including approximate and relative quantitation of the majority of identified proteins based on the emPAI value [29]. In addition, we provide a thorough insight into up- and down-regulated proteins of the S30 lysate proteome following the SOS response during cultivation of the E. coli source cells relative to standard S30 lysate, using the ICPL strategy [33]. The presented data can serve as a guideline for future protein expression strategies in view of assay development, streamlined production and fine-tuning of product quality. Addressing identified precursor degrading enzymes or proteases, as well as removing apparent bottlenecks within the enzymatic network necessary for protein biosynthesis by directed optimization, could result in increased productivity of cell-free expression systems. The report will help to render cell-free protein production a more reliable technique with higher precision, thus making the folding, assembly and quality control of newly synthesized proteins more predictable. Applications of systems biology such as rebuilding synthetic networks or designing artificial circuits by directed engineering will further profit from the precise knowledge of the S30 lysate proteome background.

Section snippets

Materials and reagents

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) or Carl Roth (Karlsruhe, Germany) unless otherwise indicated. The E. coli strain A19 (CGSC# 5997; chromosomal markers: rna-19, gdhA2, his-95, relA1, spoT1, metB1 [34]) was obtained from E. coli Genetic Stock Centre (Yale University, CT, USA). Serva ICPL Kit (Cat.# 39230.01) was used for ICPL reaction for quantitative proteomics experiments.

Cloning of apiRBP-sfGFP

An uncharacterized RNA Binding Protein (RBP) from Plasmodium vivax SaI-1 (NCBI Reference

Preparation of E. coli S30 lysates for proteome analysis

S30 lysates represent one of the most commonly used standards for cell-free expression reactions. Essential and therefore common features of almost all preparation procedures are cell harvesting at mid-log growth phase, centrifugation of the lysate at 12,000–30,000g and a so-called run-off step, which includes incubation at 37 °C to 42 °C to dissociate endogenous mRNA and ribosomes. The procedure results in sedimentation and precipitation of numerous proteins, thus fractionating the original E.

Conclusion

Our study shows that at least approximately 25% of the predicted or 40% of the expressed E. coli proteome remains in standard S30 lysates prepared for cell-free protein expression. The S30 lysate proteins have been classified and numerous data of enriched or deficient proteins match with published observations on cell-free expression studies. The S30 lysate proteome composition exhibits significant variations and may be divided into a constant core of approximately 60% of the proteins, some 16%

Author contributions

DF, EH, FB, SR, ABK contributed to conception of experimental design. EH and FB prepared the S30 lysates. EH performed protein production and biochemical analysis of expressed proteins. DF, EkH, MS performed experiments and did the data acquisition of proteomics studies. DF, EH, FB did the data analysis and interpretation. IDM and SMGM provided the apiRBP construct. EH, DF and FB wrote the manuscript. SR IDM, SMGM and CK revised the manuscript.

Acknowledgements

The authors thank Debora Teixeira Duarte for helpful discussions regarding this manuscript. This work was funded by the Collaborative Research Centre (SFB) 807 of the German Research Foundation (DFG) and by the German Ministry of Education and Science (BMBF). The software tool InfernoRDN from Pacific Northwest National Laboratory (OMICS.PNNL.GOV) is gratefully acknowledged. Further support was provided by the Andalusian Government (P11-CVI-7216 and BIO198) and the Spanish Ministry of Science

References (92)

  • R.F. Gesteland

    Isolation and characterization of ribonuclease I mutants of Escherichia coli

    J Mol Biol

    (1966)
  • E. Henrich et al.

    Screening for lipid requirements of membrane proteins by combining cell-free expression with nanodiscs

    Methods Enzymol

    (2015)
  • C. Roos et al.

    Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase

    Biochim Biophys Acta

    (2012)
  • Y. Ma et al.

    Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening

    Protein Expr Purif

    (2012)
  • F. Cymer et al.

    Mechanisms of integral membrane protein insertion and folding

    J Mol Biol

    (2015)
  • C. Berrier et al.

    Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL does not need the insertase YidC for insertion in vitro

    Biochim Biophys Acta

    (2011)
  • M. Schirle et al.

    Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry

    Mol Cell Proteomics

    (2003)
  • D. Matthies et al.

    Cell-free expression and assembly of ATP synthase

    J Mol Biol

    (2011)
  • H. Aoki et al.

    The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

    J Biol Chem

    (1997)
  • K.A. Calhoun et al.

    Total amino acid stabilization during cell-free protein synthesis reactions

    J Biotechnol

    (2006)
  • M. Ali et al.

    Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant

    J Biosci Bioeng

    (2005)
  • M.T. Smith et al.

    Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

    Process Biochem

    (2014)
  • I. Lee et al.

    Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates

    Biochim Biophys Acta

    (2008)
  • C.P. Selitrennikoff et al.

    The last two pathway-specific enzyme activities of hexosamine biosynthesis are present in Blastocladiella emersonii zoospores prior to germination

    Biochim Biophys Acta

    (1976)
  • D. Busso et al.

    Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins

    J Struct Funct Genomics

    (2004)
  • A.S. Salehi et al.

    Escherichia coli-based Cell-free Extract Development for Protein-based Cancer Therapeutic Production

    Int J Dev Biol

    (2016)
  • T. Matsuda et al.

    Cell-free synthesis of zinc-binding proteins

    J Struct Funct Genomics

    (2006)
  • C. Hein et al.

    Hydrophobic supplements in cell-free systems: designing artificial environments for membrane proteins

    Eng Life Sci

    (2014)
  • F. Löhr et al.

    An extended combinatorial 15N, 13Cα, and 13C' labeling approach to protein backbone resonance assignment

    J Biomol NMR

    (2015)
  • S. Peuker et al.

    Efficient isotope editing of proteins for site-directed vibrational spectroscopy

    J Am Chem Soc

    (2016)
  • S.H. Hong et al.

    Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation

    ACS Synth Biol

    (2014)
  • D. Proverbio et al.

    Membrane protein quality control in cell-free expression systems: tools, strategies and case studies

  • A.S. Salehi et al.

    Cell-free protein synthesis of a cytotoxic cancer therapeutic: onconase production and a just-add-water cell-free system

    Biotechnol J

    (2016)
  • G. Zubay

    In vitro synthesis of protein in microbial systems

    Annu Rev Genet

    (1973)
  • T. Kigawa et al.

    Preparation of Escherichia coli cell extract for highly productive cell-free protein expression

    J Struct Funct Genomics

    (2004)
  • W.C. Yang et al.

    Simplifying and streamlining Escherichia coli-based cell-free protein synthesis

    Biotechnol Prog

    (2012)
  • D. Schwarz et al.

    Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems

    Nat Protoc

    (2007)
  • Y. Shimizu et al.

    Cell-free translation reconstituted with purified components

    Nat Biotechnol

    (2001)
  • Y.C. Kwon et al.

    High-throughput preparation methods of crude extract for robust cell-free protein synthesis

    Sci Rep

    (2015)
  • B. Soufi et al.

    Characterization of the E: coli proteome and its modifications during growth and ethanol stress

    Front Microbiol

    (2015)
  • A. Schmidt et al.

    The quantitative and condition-dependent Escherichia coli proteome

    Nat Biotechnol

    (2016)
  • R. Aebersold et al.

    Mass spectrometry-based proteomics

    Nature

    (2003)
  • R. Aebersold

    Quantitative proteome analysis: methods and applications

    J Infect Dis

    (2003)
  • J. Rappsilber et al.

    Large-scale proteomic analysis of the human spliceosome

    Genome Res

    (2002)
  • M. Beck et al.

    The quantitative proteome of a human cell line

    Mol Syst Biol

    (2011)
  • C. Li et al.

    Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

    Sci Rep

    (2016)
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    1

    Authors contributed equally.

    2

    Present address: Chemisches und Veterinäruntersuchungsamt, Stuttgart, Germany.

    3

    Present address: Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.

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