RTS Linear Templates and Vectors
RTS Linear Template Generation Sets
Generate linear templates for RTS 100 E. coli HY reactions with RTS E. coli Linear Template Generation Sets. A Linear Template Generation Set eliminates the subcloning normally required to express proteins. It is used to add 5' and 3' T7 regulatory regions required for expression in the RTS E. coli HY Kit. The linear template obtained is ready for use in the transcription/ translation reaction:
RTS 100 E. coli Linear Template Generation Set scheme.
For the first PCR reaction, design your own gene-specific primer set that contains the necessary overlapping sequences to the T7 regulatory region DNA provided in the kit. For the second PCR reaction, the product of the first PCR is mixed with DNA fragments coding for the regulatory elements and a tag sequence. It anneals with the regulatory DNA fragments and the 3' ends are extended. This linear expression construct is further amplified via two flanking primers provided in the kit. This method avoids the time consuming step of cloning into T7 expression plasmids.
Applications
Promoter exchange
The primary application for the kit is to replace long 5' and 3' regulatory regions from most vectors with the T7 specific regions required for compatibility with RTS 100 E. coli lysates. The linear DNA provided acts as a PCR template for these regions and preparation of long PCR primers is avoided.
Tagging and Fusion
These PCR templates contain sequences that allow either N-terminal or C-terminal HA or His6 tagging or N-terminal fusion of MBP to the protein of interest. The PCR product can be used directly as a template for an RTS protein expression reaction.
RTS E. coli Linear Template Generation Set, His6-tag
The RTS E. coli Linear Template Kit, His6-tag is designed to generate linear expression templates via PCR for use in the RTS 100 E. coli HY Kit.
These PCR templates contain sequences that allow either N-terminal or C-terminal His6 tagging of the protein of interest. Additionally, the T7 specific regions required for expression in RTS 100 E. coli lysates are added.
RTS E. coli Linear Template Generation Set, HA-tag
The RTS E. coli Linear Template Kit, HA-tag is designed to generate linear expression templates via PCR for use in the RTS 100 E. coli HY Kit.
These PCR templates contain sequences that allow either N-terminal or C-terminal HA tagging of the protein of interest. Additionally, the T7 specific regions required for expression in RTS 100 E. coli lysates are added.
RTS E. coli Linear Template Generation Set, MBP fusion
The RTS E. coli Linear Template Kit, MBP fusion is designed to generate linear expression templates via PCR for use in the RTS 100 E. coli HY Kit.
The template will contain T7 regulatory elements, an N-terminal His6-tag, a Maltose Binding Protein (MBP) and a factor Xa cleavage site. MBP fusions are typically used to make a fusion partner more soluble. The His6-tag allows detection of the fusion protein, using a commonly available anti-His6 antibody. Through the factor Xa cleavage site, after proteolytic digestion the native protein of interest can be released.
RTS Wheat Germ Linear Template Generations Sets
Generate linear templates for RTS 100 Wheat Germ CECF reactions with the RTS Wheat Germ Linear Template Generation Set, His6-tag. This set eliminates the subcloning normally required to express proteins. It is used to add the T7 promoter as well as 5' and 3' regulatory untranslated regions (UTRs) required for expression in the RTS Wheat Germ lysate . The linear template obtained is ready for use in the transcription/ translation reaction:
Principle of the generation of PCR products for in vitro protein expression using RTS Linear Template Generation Sets.
As an example, the introduction of a N-terminal His6-tag is shown. Note: The 2-step PCR will result in the addition of nucleotides to both ends of the original cDNA.
In the 1st reaction, gene specific primers are used to add overlap regions to the sequence of the target gene. The product of the first PCR is mixed with two flanking primers and DNA fragments coding for the T7 promotor, eukaryotic 5’ - and 3’- regulatory elements and a His6-tag (C- or N-terminal, respectively). In the 2nd PCR Step, (the overlap extension Step) the product of the first PCR anneals with the added DNA fragments and the 5’- and 3’-ends are extended. This linear expression construct is finally amplified via the flanking primers.
Applications
Promoter exchange
The primary application for the kit is to replace long 5' and 3' regulatory regions from most vectors with the specifically optimized regions required for compatibility with RTS 100 Wheat Germ lysates. The DNA provided acts as a PCR template for these regions and preparation of long PCR primers is avoided.
Tagging and Fusion
These PCR templates contain sequences that allow either N-terminal or C-terminal His 6 tagging of the protein of interest. The PCR product can be used directly as template for an RTS 100 Wheat Germ protein expression reaction.
RTS Wheat Germ Linear Template Generation Set, His6-tag
The set allows either N-terminal or C-terminal His6 tagging of the protein of interest.
RTS E. coli Expression Vectors
RTS pIVEX His6-tag Vector Set
This set allows users of the Rapid Translation System to express their protein of interest with either an N- or C-terminal hexahistidine (His6) tag.
The vectors in the set (pIVEX2.3d and pIVEX2.4d ) contain all regulatory elements necessary for in vitro expression based on a combination of T7 RNA polymerase and E. coli cell lysates. If located N-terminally, the tag can be cleaved off by digestion with Factor Xa restriction protease (Cat.No. 1 179 888) to obtain an unmodified final product. An Anti-His6-Peroxidase conjugate (Cat.No. 1 965 085) is available for one-step detection.
The structure of the multiple cloning site allows easy switching to pIVEX vectors encoding other types of tags or fusion partners.
For detailed information of vector maps or to download the corresponding vector sequence please refer to Vector Maps.
RTS pIVEX HA-tag Vector Set
This set allows users of the Rapid Translation System to express their protein of interest with either an N- or C-terminal hemagglutinin (HA) tag.
The vectors in the set (pIVEX2.5d and pIVEX2.6d) contain all regulatory elements necessary for in vitro expression based on a combination of T7 RNA polymerase and E. coli cell lysates. If located N-terminally, the HA-tag can be cleaved off by digestion with Factor Xa restriction protease (Cat.No. 1 179 888) to obtain an unmodified final product. An Anti-HA-Peroxidase Conjugate (Cat.No. 1 667 475) is available for one-step detection. The Anti-HA Affinity Matrix (Cat. No. 1 815 016) provides a very convenient way of protein purification.
The structure of the multiple cloning site allows easy switching to pIVEX vectors encoding other types of tags or fusion partners.
For detailed information of vector maps or to download the corresponding vector sequence please refer to Vector Maps.
RTS pIVEX MBP Fusion Vector
The RTS pIVEX MBP Fusion Vector is designed for high-level expression of Maltose Binding Protein fusion proteins in the cell-free RTS E. coli system, especially for proteins which are insoluble or expressed at low yield.
The vector contains all regulatory elements necessary for in vitro expression based on a combination of T7 RNA polymerase and E. coli cell lysates. Cloning into the RTS pIVEX-MBP vector results in a fusion protein consisting of an N-terminal His6-tag, followed by MBP, a Factor Xa restriction protease cleavage site and the desired protein.
The structure of the multiple cloning site allows easy switching to pIVEX vectors encoding other types of tags or fusion partners.
For detailed information of vector maps or to download the corresponding vector sequence please refer to Vector Maps.
RTS pIVEX GST Fusion Vector
The RTS pIVEX GST Fusion Vector is designed for high-level expression of Glutathione-S-Transferase fusion proteins in the cell-free RTS E. coli system.
The vector contains all regulatory elements necessary for in vitro expression based on a combination of T7 RNA polymerase and E. coli cell lysates. Cloning into the RTS pIVEX GST Fusion Vector results in a fusion protein consisting of an N-terminal hexahistidine (His6-) tag, followed by GST, a Factor Xa restriction protease cleavage site and the protein of interest.
The structure of the multiple cloning site allows easy switching to pIVEX vectors encoding other types of tags or fusion partners.
For detailed information of vector maps or to download the corresponding vector sequence please refer to Vector Maps.
RTS Wheat Germ Expression Vectors
RTS pIVEX Wheat Germ His6-tag Vector Set
This vector set allows users of the RTS Wheat Germ platform to express their protein of interest with either an N- or C-terminal hexahistidine (His6) tag.
The vectors in the set ( pIVEX 1.3 WG and pIVEX 1.4 WG ) contain all regulatory elements necessary for in vitro expression based on a combination of T7 RNA polymerase, optimized 5´- and 3´-UTRs (untranslated regions) and wheat germ lysates. If located N-terminally, the tag can be cleaved off by digestion with Factor Xa restriction protease (Cat.No. 1 179 888) to obtain an unmodified final product. An Anti-His6-Peroxidase conjugate (Cat.No. 1 965 085) is available for one-step detection.
The structure of the multiple cloning sites allows easy switching from or to other RTS pIVEX vectors.
For detailed information of vector maps or to download the corresponding vector sequence please refer to Vector Maps.
