An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCl as the inducer

Abstract

Salt-induced overexpression of genes cloned downstream of the phage T7 10 promoter was demonstrated in an Escherichia coli strain (GJ1158) which carries a single chromosomally integrated copy of the gene for phage T7 RNA polymerase under transcriptional control of the cis-regulatory elements of the osmoresponsive proU operon. Plasmids that have been constructed to obtain overproduction of individual target gene products in strain BL21(DE3) (by addition of isopropyl-␤-D-thiogalactopyranoside as an inducer) can directly be transformed into GJ1158. The NaCl induction regimen was also shown to be associated with a decreased propensity for sequestration of overexpressed target proteins within insoluble inclusion bodies.Escherichia coli is widely used in basic research studies as a host strain for the overproduction of proteins from cloned genes, and several proteins of pharmaceutical value are also produced on a commercial scale in E. coli. In the two most commonly used strategies, a target gene is overexpressed (directly or indirectly) by temperature induction of a phage promoter or induction with isopropyl-␤-D-thio-galactopyranoside (IPTG) of the lac promoter and its variants (reviewed in reference 13).Some problems associated with temperature-induced overexpression of recombinant gene products include (i) difficulty in achieving rapid temperature upshift, particularly when handling larger culture volumes, (ii) increased likelihood of formation of insoluble inclusion bodies at higher incubation temperatures (20), and (iii) induction of several proteases in E. coli upon heat shock (4). With the lac promoter-based systems as well, the problem of inclusion body formation may be significant, in addition to the high cost and toxicity of IPTG (13).In this study, we have developed a simple, efficient, and generally applicable method for the overproduction of recombinant proteins in E. coli that makes use of NaCl as an inducer. For this purpose, we have constructed a host strain in which synthesis of the RNA polymerase (RNAP) of bacteriophage T7 has been placed under control of the osmotically inducible proU promoter of E. coli (6). Any target gene which is cloned downstream of a phage T7 promoter (e.g., 10) and introduced into this strain will be overexpressed, through a two-tier amplification strategy, upon salt addition. We also demonstrate that the propensity for inclusion body formation may be significantly reduced with the NaCl induction protocol.Methods. Most of the genetic and recombinant DNA procedures used have been described earlier (5, 19). Induction of target genes by 1 mM IPTG in derivatives of strain BL21(DE3) was done as previously described (21,22). NaCl induction experiments were done as follows. Ten-milliliter cultures were grown at 37°C to an A 600 of around 0.8 in low-osmolarity LBON medium (Luria-Bertani medium [19] with NaCl omitted, containing [per liter] 10 g of tryptone and 5 g of yeast extract [pH adjusted to 7.0 with NaOH]) supplemented with appropriate antibiotics. Each culture was then divided...

Characterization of Three Novel Adhesins of Leptospira interrogans

“…30 Escherichia coli BL21 (SI) host cells were transformed with the recombinant plasmids and grown at 30 C in LuriaBertani broth without NaCl and with 100 μg/mL of ampicillin with continuous shaking until an optical density (OD) at 600 nm of 0.6-0.8 was reached. Recombinant protein synthesis was induced for 3 hours by addition of 300 mM (rLIC11360 and rLIC11975) or 150 mM (rLIC11009) NaCl.…”

Phosphorylation of the Response Regulator CheV Is Required for Adaptation to Attractants during Bacillus subtilisChemotaxis

“…Purification of CheV and CheV D235A -pK44 and pK42 were transformed into strain OI3378 (27). 1L cultures were grown to A 600 0.7 at 37°C in LBON medium, induced by adding solid NaCl to a final concentration of 0.3 M, and grown for an additional 3 h. Purification was carried out using metal affinity chromatography according to the manufacturer's instructions (28).…”

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

“…The recombinant proteins lacking a His tag were used in the spectroscopy-based calcium-binding and denaturation assays, whereas the His-tagged LipL32 mutants were used in ECM binding assays. The LipL32 mutant plasmid constructs coding for LipL32 Q67A , LipL32 S247A , LipL32 D163-168A , LipL32 Q67A_His tag , LipL32 S247A_His tag , and LipL32 D163-168A_His tag were transformed into E. coli BL21 (SI) (19). The transformed E. coli strains were grown in 1 liter of 2YTON ampicillin until the culture optical density at 600 nm reached 0.6 at which time protein expression was induced by incubation with 300 mM NaCl for 3 h at 30°C.…”