Reconstituted Cell-free Protein Synthesis System, PURESYSTEM - Technical Note List

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in vitro Protein Synthesis Kit PURESYSTEM® Kit flyer download [PDF]

1. Procedures for protein synthesis and quick purification using PURESYSTEM kit
2. RNase and protease are little contaminated in the reagents of PURESYSTEM
3. Both eukaryotic and prokaryotic proteins can be synthesized using PURESYSTEM
4. Generation of template DNA for PURESYSTEM by 2-step PCR
5. Yield is dependent on both time and temperature in a protein synthesis reaction using PURESYSTEM
6. The reaction volume does not affect the efficiency of protein synthesis using PURESYSTEM
7. Transcript can also be used as a template for protein synthesis using PURESYSTEM
8. The template DNA containing SP6 promoter can be used for protein synthesis using PURESYSTEM

Procedures

Step 1	Addition of template DNA for start of protein synthesis reaction
Incubation at 37°C for 1 hour
Step 2	Addition of metal affinity resin for adsorption of tagged factors
Incubation at 4°C for 1 hour
Step 3	Removal of ribosome and resin with tagged factors by ultrafiltration membrane
synthesized protein can be isolated

Example of Result

Dihydrofolate reductase (DHFR; positive control) was synthesized and purified as above. Proteins in the sample at each step were detected by SDS-PAGE and CBB-staining.

Methods

Several proteins from eukaryote and prokaryote were synthesized in the presence of tRNA-Lys-BODIPY (FluoroTect™ GreenLys in vitro Translation Labelling System (Promega)) . Samples were analyzed by SDS-PAGE and Sypro®Red-staining. Bands of proteins were detected with Typhoon (Amersham Biosciences).

Result

Smmary

Protease activity contamination is very low in all tested kits, but RNase activity contamination is very high in other kits except PURESYSTEM classic kit (Figure A). Furthermore, RNase contamination in the new version kit, PURESYSTEM classic II, is about 10 % of that in classic (Figure B). These results indicate that the synthesized products (transcripts and polypepetides) are rare to degrade in the

Methods

Several proteins from eukaryote and prokaryote were synthesized in the presence of tRNA-Lys-BODIPY (FluoroTect™ GreenLys in vitro Translation Labelling System (Promega)) . Samples were analyzed by SDS-PAGE and Sypro®Red-staining. Bands of proteins were detected with Typhoon (Amersham Biosciences).

Result

Smmary

PURESYSTEM is a reconstituted in vitro translation system, which consists of E. coli translation factors. Both eukaryotic and prokaryotic proteins could be synthesized using PURESYSTEM kit. In addition, the proteins of various origins and types could be synthesized as shown in the right table.

Methods

The template DNA for synthesis of DnaJ, an E.coli protein, was generated by 2-step PCR according to the following procedure.

1. The forward and reverse primers were designed as below.

2. The reaction mixture was prepared as follows.

3. First-step PCR was performed under the following cycle condition.

4. 0.5 µl of the PCR reaction mix was applied to 1 % agarose gel (lane 1).

5. First-step PCR reaction was 100-fold diluted with H2O.

6. The second-step PCR reaction mixture was prepared as follows.

7. Second-step PCR was performed under the same cycle condition as the first step.

8. 0.5 µl of the PCR reaction was applied to 1 % agarose gel (lane 2).

Result

Reaction mixture from both 1st and 2nd PCR reactions was analyzed by agarose gel electrophoresis. In both reactions, single band was detected (indicated by arrows). The product of 2nd-step PCR was longer than that of 1st- step. This indicates that the 2nd-step product includes a regulatory region for a protein synthesis. Therefore, the 2nd-step product can be used directly as a template DNA for PURESYSTEM without further purification.

Methods

Dihydrofolate reductase (DHFR) was synthesized using PURESYSTEM at 27°C, 32°C and 37°C for 0.5, 1, 2, 3 and 4 hours. After synthesis, each reaction mixture was ultrafiltrated by Microcon YM100 (Millipore) and the flow-through fraction was collected. Samples were analyzed by SDS-PAGE and SyproOrange-staining. The bands of DHFR were detected and quantified by Typhoon (Amersham Biosciences).

Result

When DHFR was synthesized at 37°C, the protein synthesis rate was very high and the yield reached a plateau when incubated for 2 hours. On the other hand, at 32 and 27°C, the yield reached a plateau at 3 and 4 hours, respectively. In addition, the experiment also showed that as the incubation temperature was lowered, the yield decreased accordingly.

Smmary

This result indicates that PURESYSTEM works most efficiently at 37?. Therefore, we recommend for the protein synthesis reaction using PURESYSTEM to be carried out at 37?. However, some proteins have higher biological activities when synthesized at lower temperature. If your protein does not show any biological activity, please carry out the synthesis reaction at lower temperature.

The reaction volume does not affect the efficiency of protein synthesis using PURESYSTEM

Methods

Dihydrofolate reductase (DHFR) was synthesized in 50 and 1000 µl of PURESYSTEM reagents at 37 ? for 1 hour. 5 µl of each reaction was applied to SDS-PAGE. The gel was stained by SyproRed and proteins were detected by Typhoon (Amersham Biosciences).

Result

Smmary

PURESYSTEM is a reconstituted in vitro transcription/translation system, which consists of E. coli translation factors. In PURESYSTEM, the target proteins are enzymatically synthesized. The increase in the reaction volume did not alter the efficiency of protein synthseis (lanes 3 and 4), indicating that the amount of the target protein increases in proportion to the reaction volume. Therefore, it can be said that in PURESYSTEM the required volume can be easily estimated from the result of the synthesis reaction in the small volume.

Methods

The transcript of dihydrofolate reductase (DHFR) was synthesized by MEGAscript® T7 Kit (Ambion) and purified according to the manufacturer's manual. From 1, 2, 4, 10, 20 or 30 µg of transcript or 0.5 µg of plasmid DNA, DHFR protein was synthesized in 50 µl of reaction mixture using PURESYSTEM at 37? for 1 hour. After synthesis, each reaction mixture was ultrafiltrated by Microcon YM100 (Millipore) and the flow-through fraction was collected. Samples were analyzed by SDS-PAGE and SyproOrange-staining. The protein bands were detected by Typhoon (Amersham Biosciences).

Result

Smmary

PURESYSTEM is an in vitro coupled transcription/translation system, which is reconstituted by E. coli translation factors and T7 RNA polymerase. Because PURESYSTEM kit is a T7 RNA polymerase based system, the template DNA must contain T7 promoter at the upstream region of the target gene. This experiment, however, showed that transcripts can also be used as a template for the protein synthesis reaction using PURESYSTEM. Therefore, even if the target gene is located at the downstream of other promoter, protein synthesis using PURESYSTEM can be carried out from transcripts which are firstly synthesized by other in vitro transcription systems.

Methods

The template DNA for dihydrofolate reductase (DHFR) containing SP6 promoter in place of T7 promoter was generateed by 2-step PCR. From this template, DHFR protein was synthesized in the presence of SP6 RNA polymerase (Promega) in 50 µl of reaction mixture using PURESYSTEM at 37 ?for 1 hour. After synthesis, each reaction mixture was ultrafiltrated by Microcon YM100 (Millipore) and the flow-through fraction was collected. Samples were analyzed by SDS-PAGE and SyproOrange-staining. The protein bands were detected by Typhoon (Amersham Biosciences).

Result

Smmary

PURESYSTEM is an in vitro coupled transcription/translation system, which is reconstituted by E. coli translation factors and T7 RNA polymerase. Because PURESYSTEM kit is a T7 RNA polymerase based system, the template DNA must contain T7 promoter at the upstream region of the target gene. This result, however, shows that when SP6 RNA polymerase is added, template DNA containing SP6 promoter can be used for the protein synthesis reaction using PURESYSTEM. Therefore, if the target gene is located downstream of SP6 promoter, protein synthesis using PURESYSTEM can be carried out in the presence of SP6 RNA polymerase.