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PUREfrex™ Technical information and Protocol


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PURE system technology is a reconstituted cell-free protein synthesis system invented by Professor Takuya Ueda in the University of Tokyo (Ref.1, 2).

This system consists of factors for transcription, translation, aminoacylation, and energy regeneration. All proteinous factors and ribosome are highly purified individually and are assembled with substrates, such as amino acids, NTPs, and tRNAs in the buffer. Concrete factors are described in Fig.3. Translational factors includes followings; IF1, IF2, and IF3 as initiation factors, EF-Tu, EF-Ts, and EF-G as elongation factors, RF1, RF2, and RF3 as release factors, ribosome recycling factor (RRF), 20 kinds of aminoacyl-tRNA synthetase (ARS), methionyl-tRNA transformylase.

Described above, the PURE system is a reaction system consisting of a translation system only, not like other cell-free protein synthesis system using cell lysates. It indicates that the PURE system has no unknown protein and can be adjusted its composition as you like.

Fig3

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Template DNA

Fig4PCR products, circular DNA, and linear DNA are available as the template DNA for PUREfrex™. The template DNA must contain a T7 promoter and ribosome binding site (SD sequence) upstream of the gene of interest, shown in Fig. 4.

  • The gene of interest must contain an ATG initiation codon and a stop codon.
  • All stop codons, amber, ocher and opal, are available.
  • More than 10 nucleotides are necessary following the stop codon.

To generate template DNA using circular DNA, T7 terminator is necessarily following the gene of interest.

To generate template DNA using linear DNA, including PCR product and digested circular DNA by restriction enzyme, T7 terminator is not necessary in the downstream of the stop codon.

 

Fig5

To generate template DNA using PCR product, overlap extension PCR can be used by steps shown in Fig.5 with primers shown in Table 2.

Table2

 

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Protocol

The amounts given are for a 50 µL reaction. For scaling up the reaction, adjust the volume of reagents accordingly.

1. Thaw Solution I by incubation at 30Ž for 1 minute, and then cool on ice.

2. Thaw Solution II and III on ice.

3. Mix Solution I, II and III respectively by vortex and centrifuge briefly to collect each solution at the bottom.

4. Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 - 3 ng/µL per 1 kb.

Water 20-X   µL
Solution I 25   µL
Solution II 2.5   µL
Solution III 2.5   µL
Template DNA X   µL
Total 50   µL


5. Incubate the tube at 37Ž for 2 - 4 hours.

6. Analyze the synthesized products.

 

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References

1. Shimizu et al. (2001) Nat. Biotecnol., vol. 19, p. 751
2. Shimizu et al. (2005) Methods, vol. 36, p. 299

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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