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Background  Studying proteins and enzymes involved in important biological processes in the  Aedes aegypti  mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in  Escherichia coli  prove to be effective in producing milligram quantities of the expressed product. However, with the most commonly used strains having a reducing cytoplasm, soluble expression of recombinant proteases is hampered. Fortunately, new  E. coli  strains with a more oxidizing cytoplasm are now available to ensure proper folding of disulfide bonds.    Results  Utilizing an  E. coli  strain with a more oxidizing cytoplasm (SHuffle® T7, New England Biolabs) and changes in bacterial growth temperature has resulted in the soluble expression of the four most abundantly expressed  Ae. aegypti  midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). A previous attempt of solubly expressing the full-length zymogen forms of these proteases with the leader (signal) sequence and a modified pseudo propeptide with a heterologous enterokinase cleavage site led to insoluble recombinant protein expression. In combination with the more oxidizing cytoplasm, and changes in growth temperature, helped improve the solubility of the zymogen (no leader) native propeptide proteases in  E. coli . Furthermore, the approach led to autocatalytic activation of the proteases during bacterial expression and observable BApNA activity. Different time-points after bacterial growth induction were tested to determine the time at which the inactive (zymogen) species is observed to transition to the active form. This helped with the purification and isolation of only the inactive zymogen forms using Nickel affinity.    Conclusions  The difficulty in solubly expressing recombinant proteases in  E. coli  is caused by the native reducing cytoplasm. However, with bacterial strains with a more oxidizing cytoplasm, recombinant soluble expression can be achieved, but only in concert with changes in bacterial growth temperature. The method described herein should provide a facile starting point to recombinantly expressing  Ae. aegypti  mosquito proteases or proteins dependent on disulfide bonds utilizing  E. coli  as a host.    Electronic supplementary material  The online version of this article (10.1186/s12858-018-0101-0) contains supplementary material, which is available to authorized users.

Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host