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IMPACT™ Kit with T7 Express Cells |
 | | | | IMPACT™ Kit with T7 Express Cells |
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Description:
The IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) Kit utilizes engineered protein splicing elements (termed inteins) to purify recombinant proteins by a single column (Figure 1). This kit distinguishes itself from other protein fusion systems by its ability to separate a recombinant protein from the affinity tag without the use of a protease.
The IMPACT Kit allows fusion of a tag consisting of the intein and the chitin binding domain (CBD), to either the C-terminus (pTXB1) or the N-terminus (pTYB11) of the target protein (Figure 2). In the presence of thiols, such as DTT, the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag. The pTXB1 vector can also be used to express and purify a protein with a C-terminal thioester for use in Intein-mediated Protein Ligation (IPL). The IPL reaction, also referred to as expressed protein ligation, allows for the ligation of a peptide or a protein with a N-terminal cysteine to a bacterially expressed protein with a C-terminal thioester through a native peptide bond (Figure 3) for use in protein labelling and semisynthesis.
T7 Express Competent E.coli are chemically competent E. coli cells suitable for high efficiency transformation and protein expression.
1 set of this bundle includes 1X IMPACT Kit (#E6901S) and 20X transformation reactions of T7 Express Competent E. coli (#C2566H).
Advantages of T7 Express Competent E.coli: - Transformation efficiency: 2 - 6 x 108 cfu/μg
- T7 RNA Polymerase in the lac operon - no λ prophage
- Deficient in proteases Lon and OmpT
- Does not restrict methylated DNA (McrA-, McrBC-, EcoBr-m-, Mrr-)
Figure 1: Purification of Maltose Binding Protein (MBP) in a single affinity purification step: Lane 1: uninduced cell extract. Lane 2: induced cell extract showing expressed fusion protein. Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C. Marker M is the Protein Marker, Broad Range (NEB #P7702).
Figure 2: Schematic of the IMPACT System.
Figure 3: Intein-mediated Protein Ligation (IPL).
Figure 4: Transformation of a toxic mammalian clone into E. coli hosts. A T7 expression plasmid and the same plasmid containing a gene encoding a toxic mammalian protein were transformed into each host. Comparison of the relative transformation efficiencies demonstrates that the T7 Express hosts provide the levels of control necessary for transformation of potentially toxic clones. BL21(DE3) could not be transformed with the toxic clone.
Figure 5: T7-controlled expression of a non-toxic protein in E. coli hosts. A T7 expression plasmid containing a gene encoding an E. coli protein was transformed into each host, grown to 0.6 OD and induced for 3 hours. Comparison of soluble extracts from uninduced (-) and induced (+) cells shows superior control of basal expression in the T7 Express hosts while maintaining high levels of induced expression.
Notes
General notes:- Please note storage conditions for individule products.
Companion Products
IMPACT™ Kit
T7 Express Competent E. coli (High Efficiency)
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