Low Inducer Concentrations at 10°C Promotes Soluble Recombinant Expression of Aedes aegypti Mosquito Midgut Proteases in E. coli

TL;DR

Researchers found that using low IPTG concentrations and cold temperatures helps produce functional mosquito gut enzymes in bacteria.

Contribution

The study introduces a novel method using low IPTG and 10°C to improve soluble expression of Aedes aegypti midgut proteases in E. coli.

Findings

Low IPTG at 10°C slows translation, allowing proper folding of mosquito proteases.

Soluble expression levels varied with protease type and optimal growth time.

Betaine had a stronger effect at higher IPTG concentrations.

Abstract

Soluble recombinant expression of Aedes aegypti mosquito midgut proteases in Escherichia coli prove to be difficult. These enzymes depend on disulfide bond formation for structural stability. Initial attempts in BL21(DE3) were unsuccessful due to a reducing cytoplasm. The use of T7 SHuffle cells (with a more oxidizing cytoplasm) led to soluble expression. However, other factors had to be altered (use of richer media and lower (> 25°C) growth temperature). Not all mosquito proteases were equally soluble. Therefore, given the importance of IPTG in initiating transcription and translation, we set out to determine if low IPTG concentrations (3 0.1 mM) at 10°C would increase soluble production of midgut proteases. Additionally, we investigated the effect of the small molecule osmolyte betaine on the soluble expression of midgut proteases.

For this study, the focus was on Aedes aegypti Late Trypsin (AaLT), Early Trypsin (AaET), Serine Protease I (AaSPI), Serine protease V (AaSPV), and Juvenile Hormone Associated 15 (JHA15). The colder bacterial growth, along with low IPTG, slows the rate of transcription/translation of T7 RNA polymerase. Lower expression of T7 RNA polymerase, along with slower transcription activity at 10°C, prevents rapid simultaneous translation of midgut proteases. This allows recombinant proteases to properly fold. In addition, we found that different growth periods also varied among the proteases. Soluble expression for AaLT and AaET was maximal at 52 h post-induction, 72 h for JHA15, and 168 h for AaSPI and AaSPV. Surprisingly, for AaET, temperature was the only important factor. The addition of betaine to the growths had a more pronounced effect at higher (> 0.05 mM) IPTG.

Low IPTG at 10°C slows the rate of transcription/translation of recombinantly expressed mosquito proteases in bacteria. By preventing rapid accumulation in the cell, prevents aggregation, and ultimately inclusion body formation. Betaine works better at higher IPTG concentrations, but more studies are needed to better understand how this osmolyte stabilizes proteins during recombinant bacterial expression. Nonetheless, this study provides a blueprint for researchers who have never attempted IPTG concentrations > 0.1 mM to recombinantly express proteins in bacteria.

Figures