In Vitro Translation

Rosemary Jagus1, Bhavesh Joshi2, Suzzane Miyamoto3, Gregory S. Beckler4

1 Center of Marine Biotechnology and Greenebaum Cancer Center, Baltimore, Maryland, 2 Center of Marine Biotechnology, Baltimore, Maryland, 3 Molecular Hematology Branch, NHLBI, Bethesda, Maryland, 4 Promega Corporation, Madison, Wisconsin
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.2
DOI:  10.1002/0471143030.cb1102s00
Online Posting Date:  May, 2001
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Abstract

The two most frequently used systems for in vitro translation are the rabbit reticulocyte system and the wheat germ extract. These systems are useful for mRNAs isolated from cells, tissues, and capped or uncapped mRNA produced in vitro by transcription of cDNA. In a combined system, mRNA can be transcribed and translated in a single reaction. In addition these systems can be used for translation reactions with biotinylated amino acids; this allows capture of the newly synthesized protein using streptavidin immobilized on agarose.

     
 
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Table of Contents

  • Basic Protocol 1: Production an Use of mRNA‐Dependent Cell‐Free Translation System from Rabbit Reticulocytes
  • Basic Protocol 2: Production and Use of mRNA‐Dependent Cell‐Free Translation System from Wheat Germ
  • Basic Protocol 3: In Vitro Protein Synthesis in Coupled Transcripton/Translation Systems
  • Support Protocol 1: Production of Uncapped In Vitro Transcripts
  • Support Protocol 2: Production of Capped In Vitro Transcripts
  • Alternate Protocol 1: Translation of Polysomal mRNAs from Cells and Tissues
  • Alternate Protocol 2: In Vitro Translation with Binotinylated Amino Acids
  • Support Protocol 3: Capture of Biotinylated Proteins with Streptavidin‐Agarose
  • Support Protocol 4: Capture of Biotinylated Proteins with Magnetic Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Production an Use of mRNA‐Dependent Cell‐Free Translation System from Rabbit Reticulocytes

  Materials
  • Female New Zealand white rabbits, 2 to 3 kg
  • 2.5% (w/v) phenylhydrazine (see recipe)
  • 100 mg/ml ketamine hydrochloride
  • 20 mg/ml xylazine hydrochloride
  • 500 U/ml heparin in sterile water (store at −20°C)
  • 70% ethanol
  • 0.4 mg/ml sodium pentobarbital
  • Reticulocyte wash buffer I (see recipe), with and without 1 U/ml heparin (from 500 U/ml stock)
  • Reticulocyte wash buffer II (see recipe)
  • Diethylpyrocarbonate‐treated water (DEPCW; appendix 2A), sterile
  • 100 mM CaCl 2 ( appendix 2A)
  • 1 mM hemin hydrochloride (see recipe)
  • 200 U/ml creatine phosphokinase (see recipe)
  • 15,000 U/ml Staphylococcus aureus nuclease S7 (see recipe; prepare fresh)
  • 200 mM EGTA (see recipe)
  • 5 mg/ml calf liver tRNA (see recipe)
  • 2 M KCl/10 mM magnesium chloride (K/Mg; see recipe)
  • 1 mM amino acids minus methionine (see recipe)
  • [35S]Methionine, translation grade (e.g., NEN Life Sciences or Amersham)
  • 20 to 40 U/µl RNasin ribonuclease inhibitor (Promega) or equivalent
  • 1 M phosphocreatine (see recipe)
  • Translation substrate: mRNA purified from cells or tissues (see appendix 3A) or uncapped or capped in vitro transcript (see Support Protocols protocol 41 and protocol 52)
  • 5% and 10% trichloroacetic acid (TCA; see recipe)
  • 60‐ml disposable syringes
  • 1‐ml syringes
  • 60‐ml spring for 60‐ml syringes
  • No. 16 and 1.5‐in.‐long no. 20 Huberpoint needles (Fisher)
  • Liquid nitrogen
  • Glass‐fiber filters (e.g., Whatman GFC or equivalent)
  • Liquid scintillation counter and scintillation fluid

Basic Protocol 2: Production and Use of mRNA‐Dependent Cell‐Free Translation System from Wheat Germ

  Materials
  • Wheat germ
  • Wheat germ extraction buffer (see recipe; prepare fresh)
  • 100‐ml Sephadex G‐25 column (Amersham Pharmacia Biotech) preequilibrated in extraction buffer
  • 100 mM CaCl 2 ( appendix 2A)
  • 1 mM hemin hydrochloride (see recipe)
  • 200 U/ml creatine phosphokinase (see recipe)
  • 15,000 U/ml nuclease S7 (see recipe; prepare fresh)
  • 200 mM EGTA (see recipe)
  • 5 mg/ml calf liver tRNA (see recipe)
  • 50 mM ATP (see recipe for 4NTP mixture)
  • 50 mM GTP (see recipe for 4NTP mixture)
  • 50 mM and 1 M magnesium acetate
  • 20 to 40 U/µl RNasin ribonuclease inhibitor (Promega) or equivalent
  • 1 mM amino acids minus methionine (see recipe)
  • 1 M potassium acetate
  • 1 M phosphocreatine (see recipe)
  • 10 mCi/ml [35S]methionine (1000 Ci/mmol)
  • Translation substrate: mRNA purified from cells or tissues (see appendix 3A) or uncapped or capped in vitro transcript (see Support Protocols protocol 41 and protocol 52)
  • Diethylpyrocarbonate‐treated water (DEPCW; appendix 2A)
  • Sterile, RNase‐free glass powder (see recipe)

Basic Protocol 3: In Vitro Protein Synthesis in Coupled Transcripton/Translation Systems

  Materials
  • 2 M KCl/10 mM MgCl 2 (K/Mg; see recipe)
  • Amino acid mixture minus methionine (see recipe)
  • 25× transcription/translation buffer (TX/TL buffer; see recipe)
  • 10 mCi/ml [35S]methionine (1000 Ci/mmol)
  • 20 to 40 U/µl RNasin ribonuclease inhibitor (Promega) or equivalent
  • 1 M phosphocreatine (see recipe)
  • 200 U/ml creatine phosphokinase (see recipe)
  • 1 mM hemin hydrochloride (see recipe)
  • mRNA‐dependent lysate (MDL; see protocol 1, step , or see protocol 2, step )
  • Plasmid DNA, at ≥0.1 mg/ml ( appendix 3A)
  • Appropriate bacteriophage RNA polymerase (e.g., T3, T7, or SP6)
  • Diethylpyrocarbonate‐treated water (DEPCW; appendix 2A)

Support Protocol 1: Production of Uncapped In Vitro Transcripts

  Materials
  • Diethylpyrocarbonate‐treated water (DEPCW; appendix 2A), sterile
  • 5× modified transcription buffer (MTB; see recipe)
  • 12.5 mM 4NTP mix (see recipe)
  • 1 M MgCl 2 ( appendix 2A)
  • 250 mM DTT (see recipe)
  • [α‐32P]CTP (3000 Ci/mmol) in tricine, diluted in DEPCW to 10,000 to 50,000 cpm/µl
  • 20 to 40 U/µl RNasin ribonuclease inhibitor (Promega) or equivalent
  • Plasmid DNA or PCR product containing appropriate phage polymerase promoter (see appendix 3A), linearized using a restriction enzyme that generates 5′ protruding ends, at 0.2 to 0.5 mg/ml concentration
  • 20 U/µl RNA polymerase (SP6, T3, or T7; Promega)
  • 0.5 mg/ml yeast tRNA (carrier for TCA precipitation)
  • 5% trichloroacetic acid (TCA; see recipe)
  • 0.5 M EDTA ( appendix 2A)
  • RQ1 RNase‐free DNase (Promega)
  • TE buffer, pH 8 ( appendix 2A)
  • 1:1 (v/v) TE‐saturated phenol/chloroform
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 7.5 M ammonium acetate
  • 100% and 70% ethanol
  • Dry ice/ethanol bath
  • Glass‐fiber filters (e.g., Whatman GFC or equivalent)
  • Liquid scintillation counter

Support Protocol 2: Production of Capped In Vitro Transcripts

  • 5× transcription buffer (TB; see recipe)
  • 10 mM each ATP, CTP, UTP, and GTP (see recipe for 4NTP mixture)
  • 10 mM 7m(5′)Gppp(5′)G (cap analog; Amersham, Pharmacia Biotech)

Alternate Protocol 1: Translation of Polysomal mRNAs from Cells and Tissues

  Materials
  • 10× cell lysis buffer (see recipe; prepare working solution fresh before use)
  • 1 M 4‐(2‐aminoethyl)benzenesulfonyl fluoride hydrocholoride (AEBSF)
  • 8 mg/ml aprotinin
  • 2 mg/ml leupeptin
  • 250 mM DTT (see recipe)
  • 20 to 40 U/µl RNasin ribonuclease inhibitor (Promega) or equivalent
  • 0.5 M EDTA ( appendix 2A)
  • 10% Nonidet P‐40 (NP‐40)
  • Diethylpyrocarbonate‐treated water (DEPCW; appendix 2A), sterile
  • 10 mg/ml cycloheximide
  • Cells from which the polyribosomes are to be isolated
  • PBS ( appendix 2A ice‐cold liquid and frozen to a slurry
  • Sucrose buffer (see recipe; prepare fresh)
  • HEPES/KCl buffer (see recipe)
  • Beckman polyallomer thick‐walled tubes
  • Beckman TL100 centrifuge and TLA100.3 rotor
  • Additional reagents and equipment for in vitro translation (see Basic Protocols protocol 11, protocol 22, and protocol 33)

Alternate Protocol 2: In Vitro Translation with Binotinylated Amino Acids

  • mRNA‐dependent lysate (see protocol 1, step , or see protocol 2, step )
  • Biotinylated lysine‐tRNA complex (Promega's Transcend tRNA, or Boehringer Mannheim's Biotin‐Lysine‐tRNA Set) at 0.5 µg/µl (12 pmol/µl)
  • Amino acid mix, complete (see recipe)
  • Streptavidin‐alkaline phosphatase (streptavidin‐AP) or streptavidin‐horseradish peroxidase (streptavidin‐HRP)
  • Optional: Streptavidin‐linked magnetic beads (Dynal) or capture resin such as Streptavidin MagneSphere Paramagnetic Particles or Softlink Soft Release Avidin Resin (Promega), Dynabeads M‐280 Streptavidin (Dynal), or Streptavidin Magnetic Particles, Streptavidin, immobilized (gel suspension; Boehringer Mannheim)
  • Additional reagents and equipment for production and use of reticulocyte lysate (see protocol 1) or wheat germ lysate (see protocol 2) for in vitro translation, SDS‐PAGE (unit 6.1), and immunoblotting (unit 6.2)

Support Protocol 3: Capture of Biotinylated Proteins with Streptavidin‐Agarose

  Materials
  • Translation reaction mixture (see protocol 7, step )
  • Softlink Soft Release Avidin Resin (Promega), washed with 5 vol TBS at 40°C
  • TBS ( appendix 2A)
  • 2× SDS‐PAGE sample buffer (see recipe)

Support Protocol 4: Capture of Biotinylated Proteins with Magnetic Beads

  Materials
  • Translation reaction mixture (see protocol 7, step )
  • 2× biotin/streptavidin binding buffer (2× BSBB; see recipe)
  • Streptavidin‐linked magnetic beads (e.g., Dynabeads M‐280 Streptavidin, Dynal; or MagneSphere Paramagnetic Particles, Promega)
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Magnetic tube holder
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Literature Cited

Literature Cited
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