In Vitro Translation Using HeLa Extract

Gary Witherell1

1 RiboGene Inc., Hayward, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.8
DOI:  10.1002/0471143030.cb1108s06
Online Posting Date:  May, 2001
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A HeLa cell extract can be used as an alternative to rabbit reticulocyte lysate (RRL) for in vitro translation of mRNAs. It is particularly suited for synthesis of proteins that cannot be translated by RRL, is more cap dependent, has a higher capacity for faithful initiation at the correct start codon, and is more likely representative of translation in intact mammalian cells. This unit provides a detailed protocol for production and use of an mRNA‐dependent cell‐free translation system from HeLa extract. A support protocol describes production and purification of the mRNA.

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Table of Contents

  • Basic Protocol 1: Production and Use of mRNA‐Dependent Cell‐Free Translation System from HeLa Extract
  • Support Protocol 1: Production and Purification of mRNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Production and Use of mRNA‐Dependent Cell‐Free Translation System from HeLa Extract

  • HeLa S3 cells (ATCC #CCL 2.2)
  • Joklik's modified Eagle medium for suspension cultures (SMEM)
  • Heat‐inactivated iron‐supplemented calf serum (Sigma)
  • 200 mM L‐glutamine
  • 10,000 U/ml penicillin
  • 10,000 µg/ml streptomycin
  • Hypotonic lysis buffer (see recipe)
  • Dialysis buffer (see recipe)
  • Diethylpyrocarbonate‐treated H 2O (DEPCW; appendix 2A)
  • 1× phosphate‐buffered saline (PBS; appendix 2A)
  • 0.4% (w/v) trypan blue stain
  • Bradford Reagent (Bio‐Rad)
  • 1 mg/ml bovine serum albumin (BSA)
  • 10× translation mix (see recipe)
  • 0.1 M CaCl 2
  • 1 mg/ml micrococcal nuclease (Pharmacia or Fluka)
  • 0.2 M ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), pH 7.5
  • 1 M and 2 M stocks of potassium acetate (KOAc), pH 7.5
  • 10 mM magnesium acetate [Mg(OAc) 2]
  • 1 mM amino acid mix minus methionine (Promega)
  • 1 mm methionine or 35S‐labeled methionine (15 mCi/ml; >1000 Ci/mmole; e.g., NEN Life Sciences or Amersham)
  • 150 nM purified mRNA (capped or uncapped; see protocol 2)
  • 0.5, 1, and 6‐liter glass spinner flasks (autoclaved)
  • 40‐ml Wheaton Dounce homogenizer type A
  • 8‐ml collodion dialysis bags, MWCO 12 kDa (Sartorius)
  • 50‐ml plastic conical tubes
  • 30‐ml glass centrifuge tubes, one of known weight
  • 500‐ml or 1‐liter centrifuge bottles
  • Preparative centrifuge and rotor (e.g., Beckman J6‐HC centrifuge and JS‐4.2 rotor or equivalent)
  • High‐speed centrifuge and rotor (e.g., Beckman J2‐21 centrifuge and JA‐20 rotor or equivalent)
  • 2‐ml screw‐cap cryogenic vials with O‐ring seal
  • 1‐ml Wheaton Dounce homogenizer
  • 96‐well microtiter plates
  • Additional reagents and equipment for assessing cell disruption by trypan blue dye exclusion (unit 1.1) and measurement of [35S]methionine incorporation (unit 11.2)
NOTE: All solutions and equipment coming in contact with the cells or extract during the preparation must be sterile and at 4°C and aseptic technique should be used accordingly.NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Production and Purification of mRNA

  • Template: plasmid, synthetic, or PCR‐amplified DNA
  • RNase‐free DNase (optional)
  • 0.5 M ammonium acetate
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (IAA)
  • 24:1 (v/v) chloroform/IAA
  • Isopropanol
  • Diethylpyrocarbonate‐treated H 2O (DEPCW; appendix 2A)
  • 10 mg/ml ethidium bromide or Stains‐All (Kodak)
  • Microspin S‐400 columns (Pharmacia)
  • Additional reagents and equipment for production of capped and uncapped mRNAs (unit 11.2) agarose gel‐electrophoresis and spectrophotometric determination of RNA and DNA concentrations ( appendix 3A)
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Literature Cited

Literature Cited
   Belsham, G.J. and Sonenberg, N. 1996. RNA‐protein interactions in regulation of picornavirus RNA translation. Microbiol. Rev. 60:499‐511.
   Borman, A. and Jackson, R.J. 1992. Initiation of translation of human rhinovirus RNA: Mapping the internal ribosome entry site. Virology 188:685‐696.
   Carroll, R. and Lucas‐Lenard, J. 1993. Preparation of a cell‐free translation system with minimal loss of initiation factor eIF‐2/eIF‐2B activity. Anal. Biochem. 212:17‐23.
   Celma, M.L. and Ehrenfeld, E. 1975. Translation of poliovirus RNA in vitro: Detection of two different initiation sites. J.Mol. Biol. 98:761‐780.
   Dever, T.E. 1999. Translation initiation: Adept at adapting. TIBS 24:398‐403.
   Hunt, S.L., Hsuan, J.J., Totty, N., and Jackson, R.J. 1999. unr, a cellular cytoplasmic RNA‐binding protein with five cold‐shock domains, is required for internal initiation of translation of human rhinovirus RNA. Genes Dev. 13:437‐448.
   Iizuka, N. and Sarnow, P. 1997. Translation‐competent extracts from Saccharomyces cerevisiae: Effects of L‐A RNA, 5′ cap, and 3′ poly(A) tail on translational efficiency of mRNAs. Methods 11:353‐360.
   Jermutus, L., Ryabova, L.A., and Pluckthun, A. 1998. Recent advances in producing and selecting functional proteins by using cell‐free translation. Curr. Opin. Biotechnol. 9:534‐548.
   Lee, K.A. and Sonenberg, N. 1982. Inactivation of cap‐binding proteins accompanies the shut‐off of host protein synthesis by poliovirus. Proc. Natl. Acad. Sci. U.S.A. 79:3447‐3451.
   Molla, A., Paul, A.V., and Wimmer, E. 1991. Cell‐free, de novo synthesis of poliovirus. Science 254:1647‐1651.
   Rose, J.K., Trachsel, H., Leong, K., and Baltimore, D. 1978. Inhibition of translation by poliovirus: Inactivation of a specific initiation factor. Proc. Natl. Acad. Sci. U.S.A. 75:2732‐2736.
   Stiege, W. and Erdmann, V.A. 1995. The potentials of the in vitro protein biosynthesis system. J. Biotechnol. 41:81‐90.
   Svitkin, Y.V., Ovchinnikov, L.P., Dreyfuss, G., and Sonenberg, N. 1996. General RNA binding proteins render translation cap dependent. EMBO J. 15:7147‐7155.
   Villa‐Komaroff, L., McDowell, M., Baltimore, D., and Lodish, H.F. 1974. Translation of reovirus mRNA, poliovirus RNA, and bacteriophage Qβ RNA in cell‐free extracts of mammalian cells. Methods Enzymol. 30:709‐723.
   Witherell, G.W., Schultz‐Witherell, C.S., and Wimmer, E. 1995. Cis‐acting elements of the encephalomyocarditis virus internal ribosomal entry site. Virology 214:660‐663.
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