70192  STP3®, T7

Efficient transcription/translation in a single tube

The Single Tube Protein® System 3 (STP3^®) is designed for efficient in vitro production of proteins directly from supercoiled or linear DNA templates. The method is based on a linked reaction in which transcription by a bacteriophage RNA polymerase is directly followed by translation in an optimized rabbit reticulocyte lysate. Unlike other commercial kits, the patented STP3 method inherently provides an optimal amount of RNA template for translation, which results in superior protein yield and consistency between samples.

Any DNA coding sequence inserted downstream from a T7 or SP6 promoter can be quickly expressed as protein simply by adding it to the linked reaction. Suitable templates can be supercoiled or linearized plasmid DNA or PCR products. Compared with standard methods for translation of synthetic RNA templates, several tedious manipulations are avoided, including restriction enzyme digestion, linearized plasmid DNA purification, and RNA purification, which normally require four to six hours to perform. In addition, the STP3 method efficiently produces protein directly from PCR amplification products generated using appropriately designed primers. Performance can be further enhanced using the Novagen pCITE^® or pT7Blue-2 vectors containing eukaryotic translation enhancer sequences.

In the standard STP3 reaction, the DNA template (typically 0.5 µg plasmid or 2 µl chloroform-extracted PCR amplification reaction) is transcribed at 30°C for 15 minutes, followed by the addition of translation mix and continued incubation for 60 minutes. All components are premixed such that the only reagents to be added are template, water, and a choice of unlabeled methionine (included) or ³⁵S-methionine (not included).

The kit has enough reagents to perform 50 standard 50-µl reactions or 100 small-scale (25-µl) reactions. A positive control DNA containing the E. coli β-galactosidase gene is included with the kits to monitor performance. The translation product of the STP3 control reaction can be measured by standard incorporation assays, nonradioactive S•Tag™ Rapid Assay or FRETWorks™ S•Tag Assay, S•Tag™ Western Blot, or direct measurement of β-galactosidase activity with the BetaFluor™ β-Gal Assay Kit. Careful quality control ensures that the Single Tube Protein System 3 provides the highest activity, lowest background, and most consistent performance available.