Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli

Abstract

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

Keywords: Escherichia coli; Pet autotransporter platform; autotransporter; autotransporter proteins; inclusion bodies; protein secretion; recombinant protein production; secretion.

FIG 1

IrmA-S construct and secretion from E. coli BL21. (A) Crystal structure of the IrmA dimer (retrieved from PDB 5EK5). (B) Schematic diagram of the IrmA-S construct, not to scale. The irmA gene is inserted into the Pet AT platform, replacing the native Pet passenger and leaving a pelB signal sequence (SS), a 6× His tag, a linker region, the irmA gene, the autochaperone domain (AC), and the α-helix (α), the site at which the passenger is cleaved from the β-barrel. The plasmid encoding this construct was termed pET22b-IrmA-S. (C) Secretion of IrmA-S by the AT platform occurs in 5 steps, which are labeled as follows: 1, the AT platform is produced in the cytoplasm; 2, the AT platform is secreted into the periplasm by the Sec system and the PelB signal sequence is cleaved; 3, the AT platform is inserted into the outer membrane by the BAM complex; 4, the folded AT platform undergoes autoproteolysis releasing IrmA-S; and 5, IrmA-S accumulates in the culture medium. (D) The culture supernatant of E. coli BL21*DE3 dsbA::aph harboring the plasmid pET22b-IrmA-S after IPTG induction was analyzed for the presence of IrmA-S by SDS-PAGE and stained with Coomassie blue. (E) The same sample was subjected to immunoblotting using α-His antibodies. Note that although the theoretical molecular mass of IrmA-S is 29.8 kDa, it migrated in the gel with a higher apparent molecular mass.

FIG 2

Purification of IrmA-S from the culture supernatant. The culture medium of E. coli BL21*DE3 dsbA::aph harboring the plasmid pET22b-IrmA-S was harvested. The culture medium was concentrated by membrane ultrafiltration and dialyzes against the binding buffer prior to incubation with the magnetic beads for purification. IrmA-S was eluted with imidazole.

FIG 3

Comparison of IrmA-C and IrmA-S. (A) IrmA-S and IrmA-C were analyzed by SDS-PAGE with or without heating and reduction by DTT. (B) IrmA-S and IrmA-C were analyzed by differential scanning calorimetry to investigate their folding and thermal stability. Scans were recorded, and the spectra are reported in a solid line for IrmA-C and dashed line for IrmA-S. (C) IrmA-S and IrmA-C were analyzed by reverse-phase chromatography.

FIG 4

Screening of clones for IrmA-S production. Bacterial clones 2, 3, and 7 were grown in a 50 mL complex medium. IrmA-S expression was induced with IPTG after the culture reached an OD₅₉₀ of 1. (A) The growth of the clones was monitored with OD₅₉₀ measurements. For 5 h after IPTG induction, the culture medium for each clone was harvested and the proteins were precipitated in the presence of trichloroacetic acid. Precipitated protein samples were separated by SDS-PAGE and stained with Coomassie (B) or analyzed by immunoblotting with a α-His antibody (C).

FIG 5

Scaled-up production process for IrmA-S. (A) A schematic of the production process for IrmA-S. After fermentation of the isolated clone, the culture medium was separated and concentrated via tangential flow filtration (TFF). The concentrated culture medium was purified by Ni²⁺ affinity chromatography. The sample was desalted by size exclusion chromatography to remove the imidazole in the eluted fractions. Finally, the antigen was purified by ion-exchange chromatography. A culture of clone 2 was grown in a batch fermenter. IrmA-S accumulation in the culture medium was induced for 3 h with IPTG. The culture supernatant was isolated and concentrated 20-fold by tangential flow filtration. The IrmA-S was purified from the concentrated culture medium by affinity chromatography and ion-exchange chromatography. The purified antigen was analyzed by SDS-PAGE and stained with Coomassie (B) or analyzed by immunoblotting with α-His antibody (C).