Protein Synthesis Using a Reconstituted Cell‐Free System

Corinna Tuckey1, Haruichi Asahara1, Ying Zhou1, Shaorong Chong1

1 New England Biolabs, Inc, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.31
DOI:  10.1002/0471142727.mb1631s108
Online Posting Date:  October, 2014
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Abstract

Most cell‐free protein‐synthesis systems are based on cell extracts, which often contain undesirable activities. Reconstituted systems, by contrast, are composed of a defined number of purified and recombinant components with minimal nuclease and protease activities. This unit describes the use of a particular commercial reconstituted system, PURExpress. This system allows in vitro synthesis of proteins from mRNA and circular and linear DNA templates, as well as co‐translational labeling of proteins. Unique to this system, all recombinant protein components of the system are His‐tagged, allowing purification of the synthesized untagged protein by removing the rest of the system's components. Newly synthesized proteins can often be visible on an SDS‐PAGE gel and directly assayed for their functions without labeling and purification. Certain components of the system, such as ribosomes or release factors, can be omitted for specific applications. Such “delta” versions of the system are well suited for studies of bacterial translation, assays of ribosome function, incorporation of unnatural amino acids, and ribosome display of protein libraries. Curr. Protoc. Mol. Biol. 108:16.31.1‐16.31.22. © 2014 by John Wiley & Sons, Inc.

Keywords: reconstituted cell‐free protein synthesis; coupled transcription and translation; PURExpress; ribosome display; unnatural amino acid incorporation; isotope labeling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Standard Reaction Assembly for the In Vitro Transcription and Translation of Proteins Using the Purexpress Kit
  • Basic Protocol 2: Synthesis of Disulfide‐Bonded Proteins
  • Basic Protocol 3: Purification of In Vitro–Synthesized Proteins
  • Basic Protocol 4: Assay of Bacterial Ribosomes using Purexpress Δribosome Kit
  • Support Protocol 1: Preparation of Plasmid (Circular) DNA Template
  • Support Protocol 2: Preparation of Linear DNA Template by Polymerase Chain Reaction (PCR)
  • Support Protocol 3: Preparation of mRNA Template by In Vitro Transcription
  • Support Protocol 4: Synthesis and Analysis of Fluorescently Labeled Proteins
  • Support Protocol 5: Synthesis and Analysis of L‐[35S]Methionine‐Labeled Proteins
  • Support Protocol 6: Preparation of Bacterial Ribosomes
  • Support Protocol 7: Protein Synthesis Using the Purexpress Δ(aa, tRNA) Kit
  • Support Protocol 8: Protein Synthesis Using the Purexpress ΔRF123 Kit
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Standard Reaction Assembly for the In Vitro Transcription and Translation of Proteins Using the Purexpress Kit

  Materials
  • PURExpress In vitro Protein Synthesis kit (New England Biolabs, http://www.neb.com) containing:
    • Solution A (yellow tube)
    • Solution B (red tube)
    • DHFR control template
  • Murine RNase inhibitor (40 U/μl, New England Biolabs) or RNasin ribonuclease inhibitor (20 to 40 U/μl, Promega)
  • Nuclease free H 2O (Integrated DNA Technologies)
  • Template DNA (see Support Protocols protocol 51 and protocol 62) or mRNA (See protocol 7)
  • 3× SDS‐PAGE loading buffer (New England Biolabs)
  • SDS‐PAGE gel (4% to 20% Tris‐glycine; Life Technologies)
  • Nuclease‐free microcentrifuge tubes or microtiter plate
  • Air incubator set at 37°C
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and staining of gels (unit 10.6)

Basic Protocol 2: Synthesis of Disulfide‐Bonded Proteins

  Materials
  • PURExpress In vitro Protein Synthesis kit (New England Biolabs, http://www.neb.com) containing:
    • Solution A (yellow tube)
    • Solution B (red tube)
    • DHFR control template
  • PURExpress Disulfide Bond Enhancer (New England Biolabs, http://www.neb.com) containing:
    • Disulfide Bond Enhancer Solution 1
    • Disulfide Bond Enhancer Solution 2
  • Murine RNase inhibitor (40 U/μl, New England Biolabs) or RNasin Ribonuclease inhibitor (20 to 40 U/μl, Promega)
  • Nuclease‐free H 2O (Integrated DNA Technologies)
  • Template DNA (see Support Protocols protocol 51 and protocol 62) or mRNA (See protocol 7)
  • 3× SDS‐PAGE loading buffer (New England Biolabs)
  • SDS‐PAGE gel (4% to 20% Tris‐glycine; Life Technologies)
  • Microcentrifuge tubes or microtiter plate
  • Air incubator set at 37˚C
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and staining of gels (unit 10.6)

Basic Protocol 3: Purification of In Vitro–Synthesized Proteins

  Materials
  • 10 mM magnesium acetate
  • Ni‐NTA agarose (Qiagen)
  • Amicon Ultracel 0.5 ml, 100,000 MWCO spin concentrator (Millipore)
  • Bio‐Rad microspin columns (Bio‐Rad)
  • Additional reagents and equipment for setting up disulfide bonded protein–synthesis reaction ( protocol 1, steps 1 to 3)

Basic Protocol 4: Assay of Bacterial Ribosomes using Purexpress Δribosome Kit

  Materials
  • PURExpress Δribosome Kit (New England Biolabs, http://www.neb.com) containing:
    • Solution A
    • Factor Mix (essentially Solution B without ribosomes)
    • Purified E. coli ribosomes (13.3 μM, as positive control)
    • DHFR control template
  • User‐supplied bacterial ribosomes to be tested (see protocol 10)
  • 10 mM magnesium acetate (optional)
  • Murine RNase inhibitor (40 U/μl, New England Biolabs) or RNasin Ribonuclease inhibitor (20 to 40 U/μl, Promega)
  • Nuclease‐free H 2O (Integrated DNA Technologies)
  • Template DNA (see Support Protocols protocol 51 and protocol 62) or RNA ( protocol 7)
  • 3× SDS‐PAGE loading buffer (New England Biolabs)
  • SDS‐PAGE gel (4% to 20% Tris‐glycine; Life Technologies)
  • Microcentrifuge tubes or microtiter plate
  • Air incubator set at 37˚C

Support Protocol 1: Preparation of Plasmid (Circular) DNA Template

  Materials
  • E. coli culture
  • QIAprep Spin Miniprep kit (Qiagen) or other commercial plasmid purification kit
  • 10 mM Tris·Cl, pH 8.0 ( appendix 22)
  • NanoDrop (Thermo Scientific) or a similar UV spectrophotometer
  • Additional reagents and equipment for spectrophotometric ( appendix 3D) or microspectrophotometric ( appendix 3J) quantitation of nucleic acids ( appendix 3D)

Support Protocol 2: Preparation of Linear DNA Template by Polymerase Chain Reaction (PCR)

  Materials
  • Plasmid, genomic DNA or cDNA containing the protein‐coding sequence
  • Q5 High‐Fidelity 2× Master Mix (New England Biolabs) or a commercial PCR kit (e.g, AccuPrime Pfx SuperMix; Life Technologies)
  • Primers designed according to Figure  A
  • QIAquick PCR Purification Kit (Qiagen)
  • NanoDrop spectrophotometer (Thermo Scientific; also see appendix 3J)
  • Additional reagents and equipment for microspectrophotometric quantitation of nucleic acids ( appendix 3J) and agarose gel electrophoresis (unit 2.5)

Support Protocol 3: Preparation of mRNA Template by In Vitro Transcription

  Materials
  • Nuclease‐free H 2O (Integrated DNA Technologies)
  • HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs) containing:
    • NTP buffer mix
    • T7 RNA polymerase mix
    • Fluc control template
    • DNase I (RNase‐free)
    • LiCl solution
  • Linear DNA template with T7 promoter and 5′ UTR and 3′ UTR, as described in Figures and .
  • MEGAclear Transcription Clean‐up kit (Life Technologies) or other commercial kit for mRNA purification.
  • Lonza Flash Gel RNA Cassette 1.2% agarose gel (Lonza; also see unit 2.5) or Novex TBE‐Urea gels (5% to 15%; Life Technologies; also see unit 7.6)
  • Nanodrop (Thermo Scientific; also see appendix 3J)
  • Additional reagents and equipment for nucleic acid quantitation by microspectrophotometry ( appendix 3J), agarose gel electrophoresis (unit 2.5), and denaturing gel electrophoresis (unit 7.6)

Support Protocol 4: Synthesis and Analysis of Fluorescently Labeled Proteins

  Materials
  • PURExpress In vitro Protein Synthesis kit (New England Biolabs) containing:
    • Solution A (yellow tube)
    • Solution B (red tube)
    • DHFR control template
  • FluoroTectGreen Lys in vitro Translation Labeling System (Promega) containing FluoroTect Green Lys tRNA
  • Murine RNase inhibitor (40 U/μl, New England Biolabs)
  • Nuclease‐free H 2O (Integrated DNA Technologies)
  • Template DNA (see Support Protocols protocol 51 and protocol 62) or mRNA (See protocol 7)
  • 3× SDS‐PAGE loading buffer (New England Biolabs)
  • SDS‐PAGE gel (4% to 20% Tris‐glycine; Life Technologies)
  • Nuclease‐free microcentrifuge tubes or microtiter plate
  • Typhoon or other fluorescent Imaging System (GE Healthcare Life Sciences) with excitation and emission wavelengths of 532 nm and 526 nm, respectively
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2)

Support Protocol 5: Synthesis and Analysis of L‐[35S]Methionine‐Labeled Proteins

  Materials
  • PURExpress In vitro Protein Synthesis kit (New England Biolabs) containing:
    • Solution A (yellow tube)
    • Solution B (red tube)
    • DHFR control template
  • L‐[35S]Methionine (15 mCi/ml, 1000 Ci/mmol; PerkinElmer)
  • Murine RNase inhibitor (40 U/μl, New England Biolabs)
  • Nuclease‐free H 2O (Integrated DNA Technologies)
  • Template DNA (see Support Protocols protocol 51 and protocol 62) or mRNA (See protocol 7)
  • 3× SDS‐PAGE loading buffer (New England Biolabs)
  • SDS‐PAGE gel (4% to 20% Tris‐glycine; Life Technologies)
  • Nuclease‐free microcentrifuge tubes or microtiter plates
  • X‐ray film or phosphor imager ( appendix 3A)
  • Additional reagents and equipment for autoradiography or phosphor imaging ( appendix 3A)

Support Protocol 6: Preparation of Bacterial Ribosomes

  Materials
  • Bacteria growing in culture
  • Lysis buffer (see recipe)
  • Sucrose buffer (see recipe)
  • Storage buffer (see recipe)
  • Spectrophotometer
  • Centrifuge (Beckman Coulter) with JS‐4.2 and JLA‐16.25 rotors (Beckman Coulter)
  • Cell Disrupter (Constant Systems Ltd.)
  • UltraClear Centrifuge Tubes, 1 × 3½ in. (Beckman Coulter)
  • Ultracentrifuge (Beckman Coulter) with SW‐32 rotor (Beckman Coulter)
  • Slide‐A‐Lyzer, 10,000 MWCO, 3‐ to 12‐ml capacity (Thermo Scientific)
  • NanoDrop microspectrophotometer (Thermo Scientific; also see appendix 3J)
  • Additional reagents and equipment for microspectrophotometric determination of nucleic acids ( appendix 3J)

Support Protocol 7: Protein Synthesis Using the Purexpress Δ(aa, tRNA) Kit

  Materials
  • PURExpressΔ (aa, tRNA) kit (New England Biolabs) containing:
    • Solution A (minus aa, tRNA)
    • Solution B
    • Amino acid mix (control)
    • E. coli tRNA mix (control)
    • DHFR control template
  • User‐provided amino acid mixture containing desired amino acids (e.g., isotope‐labeled amino acids) at ≥3 mM concentration (lower than recommended concentrations may result in lower synthesis yields)
  • User‐provided tRNA mixture containing desired tRNAs (e.g., aminoacylated tRNAs with unnatural amino acids) at 20 mg/ml concentration (lower than recommended concentrations may result in lower synthesis yields)
  • Murine RNase inhibitor (40 U/μl, New England Biolabs)
  • Nuclease‐free H 2O (Integrated DNA Technologies)
  • Template DNA (see Support Protocols protocol 51 and protocol 62) or mRNA ( protocol 7)
  • 3× SDS‐PAGE loading buffer (New England Biolabs)
  • SDS‐PAGE gel (4% to 20% Tris‐glycine; Life Technologies)
  • Nuclease‐free microcentrifuge tubes or microtiter plate
  • Air incubator at 37°C
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and Coomassie blue staining of gels (unit 10.6)

Support Protocol 8: Protein Synthesis Using the Purexpress ΔRF123 Kit

  Materials
  • PURExpress ΔRF123 kit (New England Biolabs, http://www.neb.com) containing:
    • Solution A
    • Solution B (minus RF1, RF2, RF3)
    • Solutions of purified RF1, RF2, and RF3 (controls)
    • DHFR control template
  • Murine RNase inhibitor (40 U/μl, New England Biolabs)
  • Nuclease‐free H 2O (Integrated DNA Technologies)
  • Template DNA (see Support Protocols protocol 51 and protocol 62) or mRNA ( protocol 7)
  • Nuclease‐free microcentrifuge tubes or microtiter plate
  • Microcentrifuge
  • Air incubator at 37°C
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2) and Coomassie blue staining of gels (unit 10.6)
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Figures

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Literature Cited

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