Published: June 30th, 2023
The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.
This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.
The Gram-negative bacteria E. coli is an attractive system for recombinant protein production on both industrial and academic scale. It is not only cost-effective and straightforward to culture in batches to high densities, but a broad-spectrum of reagents, strains, tools, and promoters have been established to promote the generation of functional proteins in E. coli1. Additionally, synthetic biology techniques are now overcoming obstacles typically related to the application of post-translational modifications and folding of complex proteins2. The ability to target the secretion of recombinant proteins into culture media is attractive for improving yield and reducing manufacturing costs. Controlled packaging of user-defined proteins into membrane vesicles assists the development of products and technologies within the applied biotechnology and medical industries. Until now, there has been a lack of widely applicable methods for secreting recombinant proteins from E. coli 3.
Eastwood et al. have recently developed a peptide tagging-based method for producing and isolating recombinant protein-containing vesicles from E. coli1. This Vesicle Nucleating peptide (VNp) allows the production of extracellular bacterial membrane vesicles, into which the recombinant protein of choice can be targeted to simplify purification and storage of the target protein, and allows significantly higher yields than normally allowed from shaking flask cultures. Yields of close to 3 g of recombinant protein per liter of flask culture have been reported, with >100x higher yields than those obtained with equivalent proteins lacking the VNp tag. These recombinant protein-enriched vesicles can be rapidly purified and concentrated from the culture media and provide a stable environment for storage. This technology represents a major breakthrough in E. coli recombinant protein production. The vesicles compartmentalize toxic and disulfide-bond containing proteins in a soluble and functional form, and support the simple, efficient, and rapid purification of vesicle-packaged, functional proteins for long-term storage or direct processing1.
The major advantages this technology presents over current techniques are: (1) the applicability to a range of sizes (1 kDa to >100 kDa) and types of protein; (2) facilitating inter- and intra- protein disulfide-bond formation; (3) applicable to multiprotein complexes; (4) can be used with a range of promoters and standard lab E. coli strains; (5) the generation of yields of proteins from shaking flasks normally only seen with fermentation cultures; proteins are exported and packaged into membrane bound vesicles that (6) provide a stable environment for storage of the active soluble protein; and (7) simplifies downstream processing and protein purification. This simple and cost-effective recombinant protein tool is likely to have a positive impact on the biotechnology and medical industries, as well as discovery science.
Here, a detailed protocol, developed over several years, describes the optimal conditions to produce recombinant protein-filled vesicles from bacteria with the VNp technology. Example images of this system in practice are shown, with a fluorescent protein being expressed, allowing the presence of vesicles during different stages of the production, purification, and concentration to be visualized. Finally, guidance is provided on how to use live cell imaging to validate the production of VNp fusion-containing vesicles from the bacteria.
The bacterial work undertaken follows the local, national, and international biosafety containment regulations befitting the particular biosafety hazard level of each strain.
1. Selection of different VNps
2. Bacterial cell culture and protein induction
NOTE: Bacterial strains typically used in this protocol are either Escherichia coli BL21 (DE3) or W3110. E. coli cells are cultured in lysogeny broth (LB) (10 g/L tryptone; 10 g/L NaCl; 5 g/L yeast extract) or terrific broth (TB) (12 g/L tryptone; 24 g/L yeast extract; 4 mL/L 10% glycerol; 17 mM KH2PO4; 72 mM K2HPO4, salts autoclaved separately) media (see Table of Materials). Example images showing each step of the protein induction and subsequent isolation and purification process are shown in Figure 2.
3. Recombinant vesicle isolation
4. Soluble protein release from isolated vesicles
5. Protein concentration determination
6. Visualization of vesicle formation and isolated vesicles by fluorescence microscopy
NOTE: If the cells contain fluorescently labeled VNp fusion or membrane markers, live cell imaging can be used to follow vesicle formation. Alternatively, fluorescent lipid dyes can be used to visualize vesicles to confirm production and purification.
BL21 DE3 E. coli containing the VNp6-mNeongreen expression construct were grown to late-log phase (Figure 2A). VNp6-mNeongreen expression was induced by the addition of IPTG to the culture (20 µg/mL or 84 µM final concentration), which was subsequently left to grow overnight at 37 °C with vigorous shaking (200 rpm, ≥25 mm orbital throw). The following morning, the culture displayed mNeongreen fluorescence7 (Figure 2B), which remained visible in the media after the removal of bacterial cells by centrifugation (Figure 2C). The presence of VNp-mNeongreen within the culture and cleared culture media was confirmed by SDS-PAGE (Figure 2D). The mNeongreen-containing vesicles were isolated onto a 0.1 µm MCE filter (Figure 2E) and resuspended in PBS (Figure 2F). The purified vesicles were subsequently mounted on an agarose pad (Figure 3A-C) and imaged using widefield fluorescence microscopy (Figure 4A). The presence of vesicle membranes was confirmed using the lipophilic fluorescent dye FM4-64 (Figure 4B). The E. coli cells expressing the inner membrane protein CydB fused to mNeongreen (green) and VNp6-mCherry2 (magenta)8 show vesicle production and cargo insertion in live bacterial cells (Figure 4C). Figure 4A,B were captured using a widefield fluorescence microscope, while Figure 4C was acquired using structured illumination microscopy (SIM), using methods described previously9,10.
Figure 1: Summary of VNp technology from designing a cloning strategy to the purification and storage of extracellular vesicles. (A) Schematic of a typical VNp fusion protein. VNp at the NH2 terminus, followed by a flexible linker and an appropriate combination of affinity and fluorescence tags (Tag1, Tag 2, protease cleavage site [e.g., TEV]) and protein of interest. (B) Schematic diagram summarising the protocol for the expression and purification of recombinant protein-filled membrane vesicles from E. coli. Please click here to view a larger version of this figure.
Figure 2: Stages of production and purification of VNp6-mNg vesicles. Cultures of E. coli cells containing the VNp-mNeongreen expression construct in blue light either before (A) or after (B) IPTG-induced expression of the fusion protein. The cells from (B) were removed by centrifugation, leaving VNp-mNeongreen-filled vesicles in the media (C). (D) Equivalent samples from A, B, and C were analyzed by SDS-PAGE and coomassie staining. The vesicles were isolated onto a 0.1 µm filter (E) and subsequently washed off into an appropriate volume of buffer (F). Please click here to view a larger version of this figure.
Figure 3: Cell mounting procedure for imaging vesicles and vesicle production. (A-C) The agarose pad method and (D-F) the PEI method for mounting E. coli cells onto the coverslip. Please click here to view a larger version of this figure.
Figure 4: Microscopy images of VNp recombinant vesicles. Green (A) and red (B) emission images from different fields of FM4-64 labeled VNp6-mNeongreen-containing vesicles mounted on an agarose pad. (C) Imaging E. coli cells expressing the inner membrane protein CydB fused to mNeongreen (green) and VNp6-mCherry2 (magenta) shows vesicle production and cargo insertion in live bacterial cells. (A,B) were imaged using a widefield fluorescence microscope, while (C) was acquired using structured illumination microscopy (SIM). Scale bars: (A,B) = 10 µm; (C) = 1 µm. Please click here to view a larger version of this figure.
The amino-terminal peptide-tagged method for the production of recombinant proteins described above is a simple process, which consistently yields large amounts of protein that can be efficiently isolated and/or stored for months.
It is important to highlight the key steps in the protocol that are required for optimal use of this system. Firstly, the VNp tag1 must be located at the N-terminus, followed by the protein of interest and any appropriate tags. It is also important to avoid using antibiotics that target the peptidoglycan layer, such as ampicillin.
In terms of growth conditions, rich media (e.g., LB or TB media) and a high surface area:volume ratio is necessary to maximize vesicle production. The optimal temperature for the production of extracellular vesicles is 37 °C, but the conditions typically required for expression of the protein of interest must be considered too. For lower induction temperatures, VNp6 must be used. Crucially, induction of the T7 promoter must be achieved using no greater than 20 µg/mL (84 µM) IPTG once the cells reach an OD600 of 0.8-1.0. Proteins expressed using the system reach maximum vesicle production at either 4 h or after overnight induction.
Despite the simplicity of this protocol, it requires optimization. VNp variant fusion, expression temperatures, and induction time periods may differ depending on the protein of interest. Furthermore, there is a need to optimize the purification and subsequent concentration of extracellular vesicles from the media. The current procedure is not scalable and can be time-consuming. These are the limitations of this methodology.
The VNp technology has many advantages over traditional methods2. It allows the vesicular export of diverse proteins, with the maximum size successfully expressed to date being 175 kDa for vesicles that remain internal and 85 kDa for those that are exported. Furthermore, this technology can significantly increase the yield of recombinant proteins with a range of physical properties and activities. Exported vesicles containing the protein of interest can be isolated by simple filtration from the precleared media and can subsequently be stored, in sterile culture media or buffer, at 4 °C for several months.
The applications for this system are diverse, from discovery science to applied biotechnology and medicine (e.g., through the production of functional therapeutics)3. The ease of production, downstream processing, and high yield are all attractive qualities in these areas and especially in industry.
The authors declare no competing financial interests or other conflicts of interest.
The authors thank diverse Twitter users who raised questions about the protocol presented in the paper describing the VNp technology. Figure 1A was generated using icons from flaticon.com. This work was supported by the University of Kent and funding from the Biotechnology and Biological Sciences Research Council (BB/T008/768/1 and BB/S005544/1).
|Acros Organics (Thermofisher Scientific)
|E. coli BL21 (DE3)
|E. coli DH10β
|Filters for microscope
|Molecular Probes (Invitrogen)
|Dissolved in DMSO, stock concentration 2 mM
|Downloaded from: https://imagej.net/ij/index.html
|Isopropyl β-D-1-thiogalactopyranoside (IPTG)
|Gibco (Thermofisher Scientific)
|LED light source for micrscope
|Cairn Research Ltd
|Lysogeny Broth (LB) / LB agar
|10 g/L Tryptone; 10 g/L NaCl; 5 g/L Yeast Extract (1.5 g/L agar)
|Metamorph imaging software
|MF-Millipore Membrane filter (0.1 µm, MCE)
|Millipore Express PLUS membrane filter (0.45 µm, PES)
|Phosphate buffered saline (PBS)
|Plasmids allowing expression of protein of interest with different VNp amino terminal fusions
|Terrific Broth (TB)
|12 g/L Tryptone; 24 g/L Yeast Extract; 4 ml/L 10% glycerol; 17 mM KH2PO4 72 mM K2HPO4
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