Lucigen/Expresso® T7 Cloning and Expression System, N-His/49001-2/10 rxns

Complete Cloning and Expression System

The Expresso T7 Cloning and Protein Expression Systems are designed for fast, easy, and efficient directional cloning and expression of PCR-amplified genes. The systems are complete with pre-processed pETite™ N- or C-His cloning vectors, and two competent cell lines, supplied in single transformation vials. High efficiency HI-Control™ 10G Chemically Competent Cells enable stable cloning and HI-Control BL21(DE3) Competent Cells provide tightly controlled protein expression, thus helping you avoid expression problems seen with leaky T7 promoter-based systems. The N- or C-terminal 6xHis tagged proteins can be rapidly purified using standard Ni chromatography. Systems are also available that encode an N-terminal 6xHis SUMO tag fusion for improved solubility of expressed protein. Other Expresso Systems are available which express proteins under the control of the Rhamnose promoter.

Five-Second Directional Cloning of PCR-Amplified Genes

The Expresso T7 Cloning and Protein Expression System uses Expressioneering Technology™, an enzyme-free recombinational cloning strategy to seamlessly integrate the gene with the vector. The target gene is amplified by PCR using primers that add 18 base-pairs of vector-complementary sequence to both ends of the gene. Unlike other restriction enzyme based methods or ligase-free cloning methods, no further cleanup or enzymatic treatment of the PCR product is necessary. Simply mix 1 µl of the unpurified PCR reaction with the supplied pre-processed pETite™ T7 expression vector, and transform immediately into the HI-Control™ 10G Chemically Competent Cells provided (Figure 1). 

Cloning-Ready Vectors Save Hours of Research Time.

New pETite T7 vectors include:

1. Strong T7 promoter for high-level expression.
2. Choice of N-terminal or C-terminal 6xHis fusion tags for fast protein purification.
3. Small size (2.2kb) for easier downstream manipulation.
4. Patented CloneSmart® technology increases cloning efficiency.

High cloning efficiency with HI-Control 10G Chemically Competent Cells.

The high transformation efficiency of HI-Control 10G Chemically Competent Cells ensures recovery of clones with precise junctions and the correct orientation. For most genes, > 90% of colonies will have the target gene inserted in the correct orientation. (Figure 3).

Figure 3. High cloning efficiency with Expresso T7 System. Pre-processed pETite C-His vector was mixed with 1 µl of unpurified PCR gene product and transformed into HI-Control 10G Chemically Competent Cells. Colony PCR was performed on 18 randomly chosen colonies; 17 of 18 contained insert of the correct size.

HI-Control BL21(DE3) Cells Control Leaky Protein Expression

HI-Control BL21(DE3) Cells contain high levels of lac repressor to maintain tight control over expression of T7 RNA polymerase. Tighter control means better tolerance of potentially toxic gene products (Figure 4).

Figure 4. Expression of various proteins using the Expresso T7 System versus a pET Vector. Genes encoding a DNA polymerase, a blue fluorescent protein (BFP), or ATP synthase b subunit (membrane protein) were cloned into pET28a or pETite vectors with N-terminal or C-terminal 6xHis tags (as indicated). pET28a clones were transformed into standard BL21(DE3) cells, and pETite clones were transformed into HI-Control BL21(DE3) cells for expression. Cultures were grown in LB at 37°C to an OD600 of 0.5 to 0.7 (odd-numbered lanes) and induced for 3 hours with 1 mM IPTG (even-numbered lanes). Cells were pelleted and lysed directly in SDS-PAGE loADIng buffer, and 0.05 OD equivalents were analyzed by gel electrophoresis. The gel was stained with Coomassie blue.

Purification of Active Soluble Fluorescent Protein Using 6xHis Tag

The N- or C-terminal 6xHis tagged proteins expressed using the Expresso T7 Cloning and Expression System can be rapidly affinity-purified over commercially available nickel resins. An example of the purification of a 6xHis tagged yellow fluorescent protein cloned and expressed using the Expresso T7 System is shown in Figure 5.  

Important Product Use Information:
This product is the subject of U.S. Patent #6,709,861. Additional patent applications owned by Lucigen Corporation are pending.

The 6xHis tag is licensed from Hoffmann-La Roche, Inc., Nutley, NJ and/or Hoffmann-LaRoche Ltd., Basel, Switzerland and is provided only for the use in research.  Information about licenses for commercial use is available from Qiagen GmbH, Qiagen Strasse 1, D-40724 Hilden, Germany. Purification of the 6xHis tagged proteins with Ni-NTA manufactured by Qiagen is highly recommended for best performances and to avoid poor purification results.