A secretory system for bacterial production of high-profile protein targets

Abstract

Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.

Figure 1

Far Western blot detection of OsmY fusion proteins in culture medium samples (A) and in total expression (cell suspension) samples (B) using a Ni²⁺-NTA-HRP conjugate. Culture media were purified by Ni²⁺ chelating chromatography and analyzed by SDS-PAGE (C). Small-scale expression cultures of E. coli BL21 (DE3) pRARE R3 carrying pNIC-BASY constructs were grown in TB medium. A dominant protein band can be detected in most samples at an apparent molecular weight of around 28 kDa (black arrow). Expected molecular weights are summarized in Supporting Information Table I. Recombinant proteins detected in the culture medium and after Ni²⁺-chelating affinity chromatography purification are marked with an asterisk.

Figure 2

Far Western blot detection of OmpA and OmpF fusion proteins in culture medium samples after small-scale expression using a Ni²⁺-NTA-HRP conjugate. Cultures of E. coli BL21 (DE3) pRARE R3 carrying pNIC-BASA and pNIC-BASF constructs were grown in TB medium. The expected molecular weights are summarized in Supporting Information Table I.

Figure 3

Normalized levels of all OsmY fusion proteins that could be purified from 0.5 mL supernatant of 0.96 mL expression cultures using Ni²⁺ chelating sepharose 6FF. Percentage densitometric values were determined using the program ImageQuantTL (GE Healthcare). The average of the densitometric values of the 62, 49, 38, 28, 14 kDa bands of the SeeBlue Plus2 pre-stained marker (Invitrogen, Carlsbad) was set to be 100%. Five microliter SeeBlue Plus2 marker and 12 μL protein samples were loaded on the lanes of NuPAGE® Novex 4-12% Bis-Tris Midi gels (Invitrogen, Carlsbad) which were run in MES buffer. Constructs marked by a diamond were used for large-scale expressions.

Figure 4

OsmY fusion proteins can be isolated and purified by Ni²⁺-chelating affinity and size exclusion chromatography. Equal amounts (1 μg) recombinant fusion proteins were analyzed by SDS-PAGE. Expected and experimental masses are summarized in Table I.

Figure 5

Characterization of proteins purified from large-scale expression cultures. Target proteins are folded as shown by CD spectroscopy (A). Thermal unfolding of OsmY as measured by the change in ellipticity is reversible (B). The same OsmY-His sample was used for CD measurements at 20°C (“native”) and at 96°C (“unfolded”). Subsequently, the sample was cooled to 20°C to check for reversibility of unfolding (“refolded”). The OsmY fusion proteins are biologically active as demonstrated by surface plasmon resonance measurements (C). Both ACVR1-OsmY and monomeric NTRK2-OsmY bind their immobilized ligands BMP-6 (left diagram) and BDNF (right diagram), respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6

TEVsh protease cleavage of OsmY-NRN1 (A) and OsmY-CXCL9 (B) fusion proteins. The following samples were loaded on the lanes of NuPAGE SDS-PAGE gels (Invitrogen, Carlsbad) in (A) and (B) 1: TEVsh protease, 2: OsmY fusion protein before adding TEVsh, 3: protein mixture after TEVsh treatment, 4-6: Flow-through fractions of IMAC containing the target protein NRN1 or CXCL9 respectively, 7-9: Elution fractions of IMAC containing the OsmY-His peptide. The final protein batches were analyzed by SDS-PAGE (C). Lanes in (C) are as follows: 1-3: CXCL9, 4-6: NRN1. 3 μg (lanes 1 and 4) or 1μg (lanes 2, 3, 5, 6) of protein was analyzed. The plus and minus signs indicate the presence or absence of β-mercaptoethanol in the sample loading buffer. A mobility shift is observed for CXCL9 when analyzing the sample under nonreducing conditions.