Inducible Expression of Histidine-Tagged Ricin Toxin A Chain in E. coli

Abstract

Source: Becker, B., et al. A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast. J. Vis. Exp. (2017)

This video demonstrates the inducible expression of His-tagged ricin toxin A chain (RTA) in Escherichia coli. It outlines the steps involved in the selective culturing of bacteria transformed with a recombinant expression plasmid, induction of His-tagged RTA expression, and preparation of the bacteria for further analysis.

Protocol

1. Heterologous Expression of His-tagged Ricin Toxin A Chain (RTA) in Escherichia coli

1. Transform Escherichia coli (E. coli) cells with the expression plasmid pET24-RTA_(His)6 or the empty vector pET24a⁽⁺⁾ using standard electroporation protocols. Cells containing the empty plasmid serve as a negative control.
2. After selection of positive clones on kanamycin-containing (100 µg/mL) LB (Lysogeny Broth) plates, inoculate cells containing the RTA expression plasmid or the empty vector in 5 mL LB_kan medium (LB medium with 100 µg/mL of kanamycin) and incubate at 37 °C and 220 rpm for 24 h.
3. Supplement 1 L LB_kan medium with 5 mL pre-culture and incubate cells at 37 °C and 220 rpm until cells have reached an OD₆₀₀ of 0.8-1.0 (approximately 3-4 h). Thereafter, reduce the culture temperature to 28 °C and prepare a 1 M of isopropyl-β-D-1-thiogalactopyranoside (IPTG) stock solution in H₂O.
4. Induce RTA expression of the E. coli by adding IPTG to a final concentration of 1 mM.
5. After 3.5 h at 28 °C and 220 rpm, harvest cells by centrifugation at 10,000 x g and 4 °C for 10 min, wash the pellet two times with 5 mL of binding buffer (500 mM NaCl, 10 mM imidazole, and 20 mM KH₂PO₄, pH = 7.4) at 10,000 x g and 4 °C for 10 min, and resuspend pellet in 5 mL binding buffer.
   NOTE: Protocol can be paused at this stage, and cells can be stored at 80 °C for several days.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacterial strain
E. coli BL21 DE3(Gold)	Agilent Technologies	230130
Plasmids used in this protocol
pET24a⁽⁺⁾ (Novagen)	Millipore	69772-3
pET-RTA₍Hiₛ₆₎
Reagents
LB broth medium	Thermo Scientific	10855021	15 g agar was added for plate production
Agar	Sigma-Aldrich	05040-100G
IPTG	Sigma-Aldrich	I6758-1G