visualizing recombinant protein-filled vesicles from e. coli by fluorescence microscopy

ייצור והדמיה של שלפוחיות רקומביננטיות מלאות חלבון מאי-קולי

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.&#160;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 &#62;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 &#62;100 kDa) and types of protein; (2) facilitating inter- and intra- protein disulfide-bond formation; (3) applicable&#160;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.

מחבר זרקור: ייצור חלבונים רקומביננטיים 

ייצור וניתוח אופטימליים של בועיות מלאות חלבון רקומביננטי מאי-קולי

We are trying to understand the dynamic interactions between molecules within living cells. For biochemical analysis, it can be necessary to use large quantities of recombinant protein. These can be extremely difficult to express in a correctly-folded and functional form.If a protein was found to be misfolded or insoluble when expressed in bacteria, researchers would either have to resort to mammalian cells, which is expensive and relatively slow, or use synthetic polypeptides. Our protocol allows for high yield expression as well as export of challenging proteins from E.coli. Adding a short cleavable peptide sequence to the amino terminus of the protein not only enhances the production and yield of every protein we have tested to date, but also exports the protein from the cell into the media.Along with simplifying protein expression, it also simplifies downstream protein purification.

Watch this Scientific Journal Video about Visualizing Recombinant Protein-Filled Vesicles from E. coli by Fluorescence Microscopy at JoVE.com

Visualizing Recombinant Protein-Filled Vesicles from E. coli by Fluorescence Microscopy  

הדמיה של שלפוחיות רקומביננטיות מלאות חלבון מ- E. coli באמצעות מיקרוסקופ פלואורסצנטי

כדי להכתים את קרום השלפוחית, הוסף את צבע השומנים הפלואורסצנטי FM4-64 לשלפוחיות המטוהרות בריכוז סופי של שני מיקרומולרים ודגר במשך 10 דקות לפני ההדמיה. לאחר בידוד הבועיות מלאות החלבונים מתרבית Escherichia coli, מפיפטים את הבועיות המטוהרות על פד אגרוז עגול דק בעובי של פחות ממילימטר במשקל 2% ליברות שהותר לו להיווצר ומניחים אותו על מגלשת זכוכית נקייה. לאחר שהנוזל התייבש, הניחו מכסה בגודל 50 מ"מ על 25 מ"מ והחליקו על השלפוחיות שעל הפד.החזיקו את החלקת הכיסוי במקומה בעזרת ספייסרים וסרט הדבקה. לאחר מכן הרכיבו את המגלשה על מיקרוסקופ הפוך באמצעות מטרת טבילת שמן, והשאירו את הדגימה למשך שתיים עד שלוש דקות כדי לשקוע והטמפרטורה תתאזן. לתמונות במסגרת בודדת, השתמשו בממוצע של שלוש תמונות כדי להפחית רעשי רקע אקראיים תלויי חומרה.להדמיית קיטועי זמן, המתן שלוש עד חמש דקות בין מסגרות בודדות. הדמיית מיקרוסקופ פלואורסצנטי בשדה רחב הראתה את מי השפיר המטוהרים בצבע ירוק המכיל שלפוחיות. הצבע הפלואורסצנטי הליפופילי, FM4-64, אישר את נוכחותם של קרומי שלפוחית.מיקרוסקופיית הארה מובנית של תאי החיידקים החיים המבטאים את חלבון הממברנה הפנימית, CydB, התמזגה לירוק מי שפיר, ו- VNp6-mCherry2 הראתה ייצור שלפוחית והחדרת מטען.

visualizing-recombinant-protein-filled-vesicles-from-e-coli

Research

JoVE Journal

Biology

Visualizing Recombinant Protein-Filled Vesicles from E. coli by Fluorescence Microscopy

502 Views.  University of Kent. כדי להכתים את קרום השלפוחית, הוסף את צבע השומנים הפלואורסצנטי FM4-64 לשלפוחיות המטוהרות בריכוז סופי של שני מיקרומולרים ודגר במשך 10 דקות לפני ההדמיה. לאחר בידוד הבועיות מלאות החלבונים מתרבית Escherichia coli, מפיפטים את הבועיות המטוהרות על פד אגרוז עגול דק בעובי של פחות ממילימטר במשק...

Video: Visualizing Recombinant Protein-Filled Vesicles from E. coli by Fluorescence Microscopy  

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

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&#160;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 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 ...

Recombinant Vescicle Isolation from Bacterial Culture to Release Soluble Protein

After cloning the vesicle nucleating peptide, or VNP sequence tag, at the amino terminal of the fusion protein, culture a five-milliliter lysogeny broth, or LB starters, from fresh bacterial transformations at 37 degrees Celsius to saturation. Then, use that to inoculate 25 milliliters of terrific broth, or TB, containing appropriate antibiotic selection in a 500-milliliter conical flask. Incubate the flask in an incubator at 37 degrees Celsius while shaking at 200 rotations per minute or greater than equal to a 25-millimeter orbital throw until the culture reaches a 600-nanometer optical density value, or OD 600, of 0.8 to 1.0.Then, induce recombinant protein expression from the T7 promoter by adding IPTG to a final concentration of up to 20 micrograms per milliliter or 84 micromolar. After allowing sufficient time for induction, pellet the cells by centrifugation at 3000 G at 4 degrees Celsius for 20 minutes. Pass the supernatant through a sterile and detergent free 0.45 micron PES filter to sterilize the vesicle containing media for long-term storage.To concentrate the vesicles into a smaller volume, pass the sterile vesicle containing media through a sterile and detergent free 0.1 micron MCE filter. Then, gently wash the membrane with 0.5 to 1 milliliters of sterile phosphate-buffered saline, or PBS, and use a cell scraper or plastic spreader to carefully remove vesicles from the membrane. Transfer the vesicle concentrate to a fresh micro centrifuge tube using a pipette.Purified vesicles can be stored at four degrees Celsius. Sonicate the protein containing vesicles in the sterile media using an appropriate schedule to disrupt the vesicular lipid membranes and release protein. Centrifuge the sonicated suspension at 39, 000 G and 4 degrees Celsius for 20 minutes to remove the vesicular debris.BL21DE3 Escherichia coli containing the VNp6-mNeongreen expression construct displayed mNeongreen fluorescence being induced overnight with IPTG. The fluorescence remained visible in the culture after the removal of bacterial cells by centrifugation. The presence of VNp-mNeongreen within the culture in cleared culture media was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The resuspended purified vesicles in PBS also displayed fluorescence.

בידוד שלפוחית רקומביננטית מתרבית חיידקים לשחרור חלבון מסיס

To stain the vesicle membrane, add the fluorescent lipid dye FM4-64 to the purified vesicles at a final concentration of two micromolar and incubate for 10 minutes before imaging. After isolating the protein filled vesicles from Escherichia coli culture, pipette the purified vesicles onto a less than one millimeter thin circular 2%LB agarose pad that has been allowed to form and set on a clean glass slide. Once the liquid has dried, place a 50 millimeter by 25 millimeter cover slip over the vesicles on the pad.Hold the cover slip in place with spacers and adhesive tape. Then mount the slide onto an inverted microscope using an oil immersion objective, and leave the sample for two to three minutes to settle and the temperature to equilibrate. For single frame images, use three image averaging to reduce hardware dependent random background noise.For time-lapse imaging, allow three to five minutes between individual frames. Widefield fluorescence microscopy imaging showed the purified amnion green containing vesicles. The lipophilic fluorescent dye, FM4-64, confirmed the presence of vesicle membranes.Structured illumination microscopy of the live bacterial cells expressing the inner membrane protein, CydB, fused to amnion green, and VNp6-mCherry2 showed vesicle production and cargo insertion.