Toxic Protein Expression

Expression of heterologous proteins presents many challenges. In E. coli, expression of a non-native protein often adversely affects the viability of the host cell both during the transformation stage and during protein expression. To improve host viability and consequently improve the potential for target protein over-expression, well-regulated expression systems should be employed. In E. coli expression systems, regulation is often provided by the host cell as well as the expression vector. IPTG-inducible systems rely on the Lac repressor to bind to lac, tac, trc, T5-lac or T7-lac promoters to inhibit expression until the culture reaches an optimal density for induction. In T7 systems, the Lac repressor is also important but an even more effective means to control expression is to employ a host strain that expresses a T7 RNA polymerase inhibitor protein (e.g. LysY).

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FAQs for Toxic Protein Expression
Protocols for Toxic Protein Expression
Application Notes for Toxic Protein Expression
T7-Controlled Expression of Protein in
E. coli Hosts
A T7 expression plasmid containing a gene encoding E. coli UDG 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 expression in the T7 Express hosts while maintaining high levels of induced expression.
The Lemo System™ for Membrane Protein Expression
Lemo System™ enables simple, rapid optimization of membrane protein expression.
Lemo21(DE3) Outperforms BL21(DE3) for expression of YidC membrane protein fused to GFP
Protein expression with Lemo21(DE3) is very similar to BL21(DE3), with only a few minor changes.
Protein Expression Using the PURExpress® In Vitro Protein Synthesis Kit
25 µl reactions containing 250 ng template DNA were incubated at 37°C for 2 hours. 2.5 µl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. Note that proteins can be purified using reverse affinity chromatography (reagents not supplied). The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703)
Schematic illustration of the NEBExpress MBP Fusion and Purification System
Protein Expression using the NEBExpress MBP Fusion and Purification System

SDS-polyacrylamide gel electrophoresis of fractions from the affinity purification of MBP6-TEV-Paramyosin ΔSal. Lane 1: Protein Standard. Lane 2: uninduced cells. Lane 3: induced cells. Lane 4: purified fusion protein eluted from amylose column with maltose. Lane 5: purified protein after TEV Protease cleavage. Lane 6: target protein isolated from NEBExpress Ni Resin flow through.

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