The Champion™ pET Expression System yields the highest-level protein production in E. coli. During expression, your protein of interest can reach levels greater than 50 percent of total cellular protein. Based on T7 expression vectors originally developed by Studier and colleagues (1-3), high-level expression is achieved because the T7 RNA polymerase is more processive than native E. coliRNA polymerase and is dedicated to the transcription of your gene of interest. Protein production is further enhanced in the system by the expression strain BL21 Star™ E. coli, which significantly improves the stability of mRNA transcripts and increases protein expression up to ten-fold.
Simplified, efficient Directional TOPO® cloning allows you to quickly enter a Champion™ pET Expression vector. These kits feature linearized, topoisomerase I-activated Champion™ pET expression vectors for five-minute directional cloning. Directional TOPO® Cloning technology facilitates gene expression because:
• A proofreading enzyme is used for PCR, resulting in fewer errors in cloned genes • Greater than 90% of the clones are in the correct orientation for gene expression, reducing the time spent on colony screening
Seven Champion™ pET Directional TOPO® Expression Vectors are available (Figure 1 and Table 1): Each vector carries a T7lac promoter for high-level expression. Flexible options for simplifying protein detection, cleaving purification tags, selecting plasmid carrying clones, and/or improving protein yields are available. With the Champion™ pET Directional TOPO® vectors you can expect highest-level protein production. Figure 2 shows expression of the lacZ gene in Champion™ pET Directional TOPO® vectors. Figure 3 demonstrates efficient cleavage using TEV protease of the N-terminal tag of a β-galactosidase fusion protein expressed from pET151/D-TOPO®.
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