Using an E. coli Type 1 secretion system to secrete the mammalian, intracellular protein IFABP in its active form

A biotechnological production of proteins through protein secretion systems might be superior to the conventional cytoplasmic production, because of the absence of large amounts of proteases present in the extracellular space and the ease of purification or downstream processing. However, secretion of proteins is still a trial-and-error approach and many proteins fail to be secreted. Recently, a study of a Type 1 secretion system revealed that the folding rate of the passenger protein dictates secretion efficiency. Here, the well-known MalE failed to be secreted when fused to a C-terminal fragment of the natural substrate haemolysin A. In contrast, slow-folding mutants of MalE were secreted in high yields. However, MalE is a bacterial protein that is targeted to the periplasmic space of E. coli and possesses the intrinsic capability to cross a membrane. Therefore, we applied the same approach for another eukaryotic protein that resides in the cytoplasm. As an example, we chose the intestinal fatty acid binding protein (IFABP) and highlight the universal potential of this Type 1 secretion system to secrete proteins with slow-folding kinetics (here the G121V mutant). Finally, a one-step purification protocol was established yielding 1 mg of pure IFABP G121V per liter culture supernatant. Moreover, secreted IFABP G121V was shown to reach a folded state, which is biologically active.

► We examined the ability of a bacterial T1SS to secrete a eukaryotic, cytoplasmic protein.
► A slow-folding mutant of the protein was secreted in good yields.
► An effective one-step purification protocol to obtain pure protein was established.
► The protein was correctly folded and active after being secreted with our system.