A generalised module for the selective extracellular accumulation of recombinant proteins

Abstract

Background: It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu.

Results: Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems.

Conclusions: The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.

Figure 1

AT-mediated accumulation of heterologous proteins in the culture medium. (A) Schematic diagram of Pet fusion constructs. Heterologous protein insertions in the Pet passenger domain are shown by dark boxes marked HP or with the name of the protein, and are also listed on the right. Abbreviations BB and BP on the left refer to the type of protein fusion generated by insertion of foreign DNA into the pet gene between the restriction sites BglII and BstBI or BglII and PstI, respectively. The co-ordinates above the figure are given for the amino acids derived from the de novo synthesised pet gene. The positions of these sites in the context of the quaternary structure are depicted in Additional file 1: Figure S1. The arrow at position 1018 denotes the cleavage site in the α-helix that effects release of the passenger domain into the culture medium. Modification of this site results in surface display of molecules (Figure 2). The abbreviations SS, AC and α denote the positions of the signal sequence, autochaperone domain and α-helix, respectively. (B) The presence of secreted heterologous proteins in the culture medium was detected by SDS-PAGE or immunoblotting with anti-Pet. Equivalent volumes of medium were analysed. The structures of several heterologous proteins are depicted (not to scale). (C) Investigation of the folded state of secreted heterologous fusion proteins. Far-UV CD spectra of Pet and several heterologous proteins are shown in millidegrees (mdeg). mCherry-Pet-BP harvested from the culture supernatant is shown.

Figure 2

Monitoring cellular integrity of theE. colihost strain expressing AT chimeras. (A) SDS-PAGE analysis of OM (M) and culture supernatant (S) fractions derived from E. coli TOP10 expressing Pet*, mCherry*and ESAT6*. Non cleaved species are denoted by arrows. Molecular weight markers (MWM, kDa) are indicated to the right of the panel. OM fractions demonstrating the presence and absence of the β-domain are shown in Additional file 6: Figure S5. (B)E. coli cells expressing empty vector, Pet*, mCherry*, ESAT6* and their cleaved parents were harvested 2 h after induction and subjected to indirect immunofluorescence using the indicated antibody. For each population expressing a cleavage deficient variant, a sample was divided in two: one half was probed with an antibody to the periplasmic protein BamD, while the other half was permeabilised (−P) and subsequently probed with the same antibody. Corresponding fields are also shown by phase contrast microscopy. For the mCherry constructs panels showing mCherry derived fluorescence are shown. (C) The integrity of E. coli host cells expressing wild type Pet and secreted ESAT6-Pet fusions were assessed by staining with BOX and PI prior to and 2 h post induction. Q3 represents the population that is viable and healthy and did not label with either stain. Q2 represents cells that stain with both stains and are no longer viable. Q4 (BOX-positive) represents cells with impaired membrane potential suggesting compromised membrane integrity. (D) Periplasmic leakage from E. coli TOP10 cultures secreting Pet or ESAT6-Pet fusion proteins was assessed by measuring (2 h after induction) the activity in the culture medium of the periplasmic enzyme alkaline phosphatase. Clarified whole cell lysate was used as a positive control and the wild-type plasmid-free strain as a negative control. There is no significant difference between the negative control and culture medium derived from strains expressing Pet or ESAT6-Pet-BB. In contrast, culture medium from both constructs displayed activity significantly less than the positive control. Absorbance measurements are derived from equal volumes of culture and are normalised relative to positive control (in %). The error bars represent standard error for two independent data sets.

Figure 3

Modification of the Pet-AT secretion platform. (A) SapA, Pmp17, Pet and a Pet derivative (PetΔD1) lacking the serine protease domain were modified to add a His₆-Tag to the N-terminus of the secreted passenger domain. The His-tagged Pmp17 protein was expressed in an E. coli TOP10 dsbA strain and the rest of proteins were produced in the wild-type E. coli TOP10. In all cases the proteins are well secreted. The cysteine-containing Pmp17 was expressed in E. coli TOP10 (wt) and a dsbA^- derivative. A full length protein is present in the E. coli TOP10 dsbA^- derivative but not the E. coli TOP10 parent strain. Break down products are apparent and correspond to proteins with a truncated N-terminus. A multicomponent construct was created by fusing DNA encoding Ag85 to ESAT-6 and Pet (see Figure 1A) to encode a single polypeptide chain contiguous with the AT-translocation unit. This latter chimera was detected in the culture supernatant with antibodies directed at Pet and ESAT-6. Equivalent amounts of culture supernatant fractions were analysed by SDS-PAGE. (B) Secretion of heterologous fusions from S. Typhimurium. Culture medium from S. enterica SL1344 strains expressing ESAT6-Pet-BP and ESAT6-PetΔ*6 (see Figure 4) were harvested and analysed by SDS-PAGE and detected by immunoblotting with a polyclonal antibody to ESAT-6. In all SDS-PAGE gels the positions of the molecular weight markers (MWM, kDa) are depicted at the right side of the panel. The equivalent OM fractions demonstrating the presence of the cleaved β-barrel are shown in Additional file 5: Figure S4.

Figure 4

Identification of the minimal AT module permitting secretion of heterologous proteins to the culture supernatant fraction. (A) Schematic of ESAT6-Pet-BP protein fusion and some truncations created to determine the minimal C-terminal Pet fragment capable of ESAT-6 secretion. The Δ*1, Δ*2, Δ*6, Δ*17 and Δ*20 Pet truncations are shown while for simplicity the intermediate variants Δ*3–Δ*5, Δ*7–Δ*16, Δ*18 and Δ*19 are omitted. Abbreviations are the same as in Figure 1. (B and C) Detection by western immunoblotting of ESAT6-Pet (B) and ESAT6-Pic (C) chimeras expressed in E. coli TOP10. The TCA-precipitated culture supernatants were analysed by SDS-PAGE and probed with polyclonal anti-ESAT6. The equivalent OM fractions demonstrating the presence of the cleaved β-barrel in the OM are shown in Additional file 6: Figure S5.