High-yield vesicle-packaged recombinant protein production from E. coli

Abstract

We describe an innovative system that exports diverse recombinant proteins in membrane-bound vesicles from E. coli. These recombinant vesicles compartmentalize proteins within a micro-environment that enables production of otherwise challenging insoluble, toxic, or disulfide-bond containing proteins from bacteria. The release of vesicle-packaged proteins supports isolation from the culture and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine.

Keywords: disulfide bond; extracellular vesicles; recombinant protein.

Graphical abstract

Figure 1

Recombinant vesicle formation (A) SIM fluorescence images of E. coli expressing αSyn-mNeongreen (green) and OmpA-mCherry (red) show production of extracellular αSyn-containing membrane vesicles. (B–D) OmpA-mCherry SIM fluorescence (B) and TEM (C) and (D) images illustrating that VNp induces membrane curvature in E. coli. (E) EM of vesicles generated from VNp-expressing E. coli cells that were cultured on prepared grids. (F) mCherry (magenta) and mNeongreen (green) SIM fluorescence of VNp-mNeongreen OmpA-mCherry-expressing E. coli cells. (G) Anti-mNeongreen immuno-EM of a section through E. coli associated VNp-mNeongreen induced vesicle. (H) TEM images of isolated VNp-mNeongreen-containing vesicles. (I) Coomassie stained gel of cell culture and filtered media of VNp-mNeongreen-expressing cells. (J) Schematic of VNp-induced cargo-containing vesicles. (K) Coomassie stained samples of uninduced and induced cultures or filtered induced cultures of VNp-DARP-, VNp-uricase-, and VNp-stefin A-expressing cells. (L and M) Average soluble yields per liter of culture derived from cell extracts (empty boxes) or filtered culture media (filled boxes) for each recombinant protein examined. Recombinant proteins lacked (L) or possessed (M) a fluorescent mNeongreen fusion. Errors are SD from ≥3 experimental repeats.

Figure 2

VNp allows expression of functional disulfide-bond-containing IgG1 fusion dimers (A and B) Conventionally stained (A) and anti-mNeongreen immuno-stained (B) EM serial section images of VNp-mNeongreen-Etanercerpt-induced inward membrane curvature in E. coli. (C) Schematic of the VNp-mNeongreen-TEV-etanercept fusion protein. (D and E) Anti-mNeongreen western blots illustrating disulfide-bond-dependent ooligomerization (D) and protein A-binding IgG1 functionality (E) of VNp-mNG-etanercept purified from E. coli. (D) VNp-mNG-etanercept disulfide-bond-dependent oligomers (∗) are disrupted by the addition of the disulfide-bond-disrupting reducing agent, DTT. (E) VNp-mNG-etanercept-His₆ fusion was affinity purified from E. coli and bound to protein A Dynabeads. Beads were subsequently washed in binding buffer before being boiled in SDS-PAGE loading buffer to release bound proteins. Predicted size of VNp-mNeongreen-etanercept: 83.9 kDa. (F) Anti-His western blot of wash (unbound) and protein A-bound fractions of TEV cleaved VNp-mNeongreen-TEV-etanercept-His fusion mixed with protein A Dynabeads. Predicted size of etanercept: 52.5 kDa. This illustrates that unlabeled etanercept remains soluble and functional upon removal of the VNp-mNeongreen tag.

Figure 3

VNp dimers produce VNp-fusion-containing cellular membrane packages (A) Size-exclusion chromatography profiles of purified recombinant VNp-mNG and VNp-LZ-mNG proteins (inset) confirmed that introduction of a leucine zipper (LZ) motif to the VNp-mNeongreen (mNG) fusion induced stable dimer formation. Each fusion protein as well as protein standards (29 kDa carbonic anhydrase: blue; 66 kDa BSA: red; 443 kDa apoferritin complex: yellow) were run using identical conditions. Whereas the VNp-mNG (black) elution profile was consistent with a monomeric protein, the VNp-LZ-mNG (gray) eluted from the column in earlier fractions consistent with it existing predominantly as a dimer. (B–D) Anti-mNeongreen immuno-EM images of sections though E. coli expressing VNp-LZ-mNeongreen (B) and (C) and SIM images of CydAB-mNeongreen labeled inner membranes in E. coli expressing VNp-LZ (D) show that the VNp-LZ dimer concentrates within the lumen of cytosolic inner membrane-bound vesicles.