SpeedRead Lysates

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SpeedRead Lysates
2003. 6. 10 ???
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Conventional translation vs. SpeedRead
reaction
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SpeedRead Lysate 1

• uncapped in vitro transcripts, such as those
  produced with T7, SP6 and T3 RNA polymerases.
• permissive translation initiation
• initiation at secondary sites may occur
• however, the majority of product is observed
  as full length protein.

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SpeedRead Lysate 2

• RNA containing 5' cap structures or the EMCV
  (encephalomyocarditis) internal ribosome entry
  site found in Novagens pCITE vectors.
• stringent translation initiation conditions
• optimal amounts of full length proteins.

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(No Transcript)
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DNA Templates for Preparing in vitro Transcripts

• Suitable templates for in vitro transcription
• circular, linear plasmids and PCR products
• It is also useful to include an optimal
  translation initiation sequence as part of the
  primer immediately preceeding the 5' end of the
  target sequence.

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Optimal performance

• Cloned downstream from the translation enhancer
  present in pCITE vectors.
• -gt EMCV
• an internal entry point for initiation
• of translation by eukaryotic ribosomes
• This CITE dramatically increases (10-fold) the in
  vitro translation efficiency.

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pCITE vectors

• The pCITE vectors are designed for dramatic
  enhancement of the efficiency of in vitro
  translation of cloned sequences.
• The pCITE-4ac() vectors also contain an
  optional N-terminal STag sequence
• gt Nonradioactive assay
• Western blot analysis
• affinity purification

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pCITE-2a-c() Vectors
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pCITE-4a-c() Vectors
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Translation

• 1050 µg RNA/ml
• (100300 µg/ml if total RNA is used)
• Always include a blank reaction without added
  RNA
• Control reaction with control RNA

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Translation Reaction

• 20 µl SpeedRead Translation Mix
• 1 µl 625 µM Met (non-radioactive reactions)
• or 2 µl 35S-Met in aqueous buffer (about 20 µCi)
• x µl RNA sample (free of salts)
• y µl RNase-free water
• 25 µl Total volume
• Mix by stirring with the pipet tip and incubate
  at 30 C for 60 min.

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Analysis of Translation Products

• Incorporation Assay (35S-methionine label)
• Gel Analysis
• STag Nonradioactive Assays and Affinity
  Purification

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STag

• The STag rapid assay is based on the
  reconstitution of ribonucleolytic (RNase S)
  activity.
• Activity is measured by reading the absorbance of
  the supernatant at 280nm.

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Advantage

• for rapid and convenient in vitro translation of
  RNA transcripts.
• only the template RNA and 35S-Met label (or
  unlabeled methionine) need to be added.

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Disadvantage

• Not for other translation conditions, such as
  labeling with amino acids other than Met or salt
  optimization
• the need to prepare and purify RNA templates
  separately.