Vmax™ Express Competent Cells

Recombinant Protein Expression: Fast Growth and High Yields of Soluble Protein

Vmax™ Express is a novel, fast-growing bacterial strain designed and optimized for high-level recombinant protein expression. This rationally engineered, next-generation prokaryotic protein expression system can serve as a replacement for slow growing E. coli systems that are prone to low yields and the expression of proteins as insoluble inclusion bodies. Vmax™ cells are derived from the marine microorganism, Vibrio natriegens. This gram-negative, non-pathogenic bacterium has a doubling time twice that of E. coli and robust transcription and translation systems that support this rapid growth rate. This means that Vmax™ Express can produce greater amounts of biomass and generate large amounts of protein in a shorter time period.

With a protein expression workflow similar to E. coli, Vmax™ Express is compatible with plasmids and antibiotics commonly used with bacterial expression systems such as E. coli BL21(DE3). The Vmax™ Express workflow, fast growth, and ability to express a wide range of proteins make it ideal for routine protein expression. Additionally, Vmax™ Express can help overcome common recombinant protein expression challenges such as low yields or the expression of insoluble, or inactive proteins. Built with a tightly controlled, IPTG-inducible T7 promoter system, Vmax™ Express cells can be cultured using routine growth medium, as well as commercial auto-induction media, or our Vmax™ Enriched Growth Media (recommended for rapid growth and greatest accumulation of biomass).

In contrast to E. coli, Vmax™ Express cells grow rapidly at both 30°C and 37°C and tolerate the induction of protein expression over a wide range of starting ODs and IPTG concentrations. In addition, Vmax™ Express cells can be incubated for up to 24 hours post induction to maximize the amount of soluble protein produced. This flexible protein expression workflow and rapid doubling time allows you to go from transformed cells or a glycerol stock to a large-scale culture ready for analysis or protein purification in as few as three standard workdays compared to four days with E. coli.

Learn more about Vmax Express - Watch the 2017 PepTalk Presentation

Summary:

  • Fastest growth rate of any known bacterial host with a doubling time of ~14 minutes
  • Produces up to twice the biomass of E. coli with up to 4X the protein
  • Enables expression of high levels of soluble recombinant proteins
  • Tightly regulated, IPTG-inducible T7 promoter system
  • Compatible with commonly used E. coli expression vectors, antibiotics, and growth media
  • Protein expression workflow similar to that of E. coli
  • Tolerates induction over wide range OD’s (0.3 to 1.0) and IPTG concentrations (0.5 to 1.0 mM)
  • Efficient protein expression up to 24-hours post-induction
  • Rapid results - Go from cells to large-scale culture ready for protein purification in three days

E. coli and Vmax™ Express Comparison

Protein Expression Strain Performance Attribute E. Coli Strains Vmax™ Express
Compatible with standard plasmid origins of replication (e.g. pMB1, ColE1, pUC, p15A)
Able to use common inexpensive growth media
Plasmid selection uses common antibiotic resistance markers (Amp, Tet, Kan, Cm)
Tightly controlled IPTG Inducible T7 transcription system
Doubling time of ~14 minutes
Biomass >14 OD after 24 hours growth
Flexibility to induce expression at range of OD600
Rapid growth at both 30° and 37°C*
Culture ready for protein purification and analysis in 3 days
*Optimum incubation temperature for Vmax™ Express in liquid media is 30°C. However, the Vmax™ Express growth rate at 37°C exceeds that of E. coli.

Recombinant Protein Expression with Vmax™ Express is 1 Day Faster than E. coli

Comparison of Vmax™ Express Rapid Workflow with E. coli Protein Expression Workflow. Vmax™ Express utilizes a workflow similar to E. coli for protein expression. However, the rapid growth rate of Vmax™ Express allows multiple steps of the protein expression workflow to be completed in one standard workday instead of over multiple days.

Vmax™ Express is Compatible with Typical Media, Plasmids, Antibiotics Used for E. coli

A variety of media commonly used for E. coli also support the growth of Vmax™ Express. However, Vmax™ Express grows most rapidly in media with higher salt concentrations. For some commonly used media, salt supplements may allow for more optimal growth. The table below outlines some of the recommended media that allow for robust growth. For the fastest doubling times and best growth at a variety of temperatures, we recommend Vmax™ Enriched Growth Media (SGI-DNA catalog number CL1500-1000).

Vmax™ Express Compatibilities

Media Format 25°C 30°C 37°C Recommendation
Vmax™ Enriched Growth MediaLiquid CultureGrowthGrowthGrowth++++
Preferred
Enhanced 2xYT MediaLiquid CultureGrowthGrowthGrowth+++ Recommended
Brain Heart Infusion Broth + v2 salts‡Liquid CultureGrowthGrowthGrowth++
Recommended
LB-Miller† Liquid CultureGrowthGrowthSupplement with v2 salt‡++
Terrific broth Liquid CultureNot testedGrowthGrowth+
MagicMedia™Liquid CultureNot testedGrowthGrowth+
LB-Miller†Agar PlateSlow GrowthGrowthGrowth+++ Recommended
Brain Heart Infusion agar + v2 salts‡Agar PlateGrowthGrowthGrowth++
*Recommendation Key: ++++ = Preferred media, +++ = Recommended, ++ = Acceptable, + = Acceptable,but other media may support better growth
†LB-Miller media contains 10 g/L of NaCl. Other LB variants (e.g. LB-Lennox and LB-Luria) contain less salt and will not support optimal growth.
‡v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2 (these are in addition to salts present in base media)

Vmax™ Express is Compatible with Many Plasmid Origins of Replication

A variety of commonly used plasmid origins of replication and antibiotic selection markers are compatible with Vmax™ Express. This allows the use of existing expression constructs for recombinant protein expression. After transformation or recovery from a glycerol stock, Vmax™ Express cells can be plated on nutrient agar with appropriate antibiotics. The tables below provide the growth conditions and suggested antibiotic concentrations for both solid and liquid media.

Vmax™ Express Compatible Origins of Replication

Plasmid Origin of Replication Plasmid Backbone Growth Plate Recommendation
pMB1 pBR322 pET vectors Recover at 30 or 37°C
(37°C recommended)
ColE1 pCDNA3.0
pGEM5zf
Recover at 30 or 37°C
(37°C recommended)
pUC pUC19 If using Kan selection, recover at 30°C only.
p15A pACYC184 Recover at 30 or 37°C
(37°C recommended)

Antibiotic Concentrations for Maintaining Antibiotic Selection in Vmax™ Express

Antibiotic Marker Concentration (µg/mL) for Solid Media Concentration (µg/mL) for Liquid Culture
Ampicillin/Carbenicillin 50 50
Kanamycin 100 50-100
Tetracycline 1 1
Chloramphenicol 5-12.5 12.5-25

Applications

  • Lab scale expression of recombinant proteins
  • Large scale expression of recombinant proteins
  • Expression of proteins that express at low levels or as inclusion bodies in E. coli
  • Expression of active proteins

Performance Data

Vmax™ Express Has the Fastest Growth Rate and Generates Greater Biomass

Figure 1. Growth comparison of Vmax™ with other E. coli strains. Vmax Express™ demonstrates faster growth and a greater accumulation of biomass as compared to other common laboratory strains. Panel A. Overnight cultures of the indicated strains grown in Brain Heart Infusion Broth + v2 salts were inoculated to an OD600 of 0.03 and grown at the E. coli optimum of 37°C. At the indicated times, the OD600 of each culture was determined. Panel B. LB-Miller plates agar plates were streaked with the indicated strains and incubated at 37°C. Images of the growth were recorded at the times indicated.

Vmax™ Express Generates Larger Amounts of Soluble Protein


Figure 2. Expression of Green Fluorescent Protein (GFP) in Vmax.
To evaluate expression levels in Vmax™ Express, a plasmid expressing Green Fluorescent Protein (GFP), was introduced to cells and induced with IPTG at an OD of 0.5. Except where indicated in the figure, expression was performed at 30°C. At the time points indicated, samples of IPTG induced (I) and uninduced (U) were taken for analysis. Panel A. UV light was used to visualize active GFP in equal volumes of each cell lysate. In in Vmax Express cells, detectable signal was observed in all induced samples. Panel B. Lysates from panel A were examined by SDS-PAGE followed by Coomassie Blue staining to visualize the amounts of expressed 27 kDa GFP protein. Consistent with the activity assay from panel A, Vmax Express demonstrated a consistent increase in the amount of protein expressed in IPTG-induced cultures.

Figure 3. Expression of Metabolic Protein Aldose 1-Epimerase in Vmax™ Express and E. coli.
A plasmid expressing Aldose 1-epimerase was transformed into the Vmax Express™ strain and E. coli strain BL21(DE3). A single colony was picked and used to generate a seed culture for lab scale protein expression. Cells were induced with 1 mM IPTG at an OD of 0.5 and samples were taken at 4 and 24 hours post induction. Equal volumes of each sample were lysed and insoluble material removed by centrifugation. The soluble protein was analyzed by SDS-PAGE and Coomassie Blue staining. The resulting data demonstrate that Vmax results in larger amounts of soluble Aldose 1-epimerase protein at 24 hours as compared to BL21(DE3).

Reliable Protein Expression over a Range of OD600 Readings and IPTG Concentrations


Figure 4. Expression of Recombinant Proteins with Vmax at Different Densities and IPTG Concentrations. Vmax Express Cells containing an expression construct for uronate dehydrogenase were grown in enriched 2xYT medium at 30°C to variable OD and induced with 0.5, 1, and 2 mM IPTG and grown for an additional 24 hours. Cells were collected, lysed, and soluble protein recovered for SDS-PAGE followed by Coomassie staining. Migration of 30 kDa uronate dehydrogenase recombinant protein is indicated by green arrow. Under all conditions tested, Vmax Express generated similar biomass and significant amounts of soluble protein per cell.

Vmax™ Express Outperforms E. coli BL21 for the Expression of a Variety of Proteins

Protein Size (kDa) BL21 (DE3) Vmax Express Comments
Type I PQQ Dehydrogenase 69 Insoluble in BL21. Soluble in Vmax up to 24 hr post-induction.
DHG Dehydrogenase 25 Protein expressed in BL21. Minimal protein detected in Vmax.
Alditol Oxidase 41 Expressed in both. More protein and biomass using Vmax. .
GFP 27 Expressed in both. More protein and biomass using Vmax.
Glucose Dehydrogenase 40 Expressed in both. More protein and biomass using Vmax.
Uronate Dehydrogenase 30 Insoluble in BL21. Soluble in Vmax up to 24 hr post-induction.
Comparison of Expression of Various Proteins Using Vmax Express and E. coli BL21(DE3). E. coli BL21(DE3) cells or Vmax Express™ cells containing expression constructs for the proteins listed in the table above were used for recombinant protein expression. In all cases expression was induced using 1 mM IPTG at an OD of 0.5. After induction, cells were incubated for 4 and 24 hours. BL21 cells and Vmax Express cells were grown at their optimum temperature of 37ºC and 30ºC respectively. Protein expression was evaluated relative to an uninduced sample using SDS-PAGE stained with Coomassie Blue.

Vmax™ Express Electrocompetent Cells

Description Catalog # Reactions* Price
Vmax™ Express Electrocompetent Cells CL1100-05 5 $67
Vmax™ Express Electrocompetent Cells CL1100-10 10 $127
Vmax™ Express Electrocompetent Cells CL1100-20 20 $237
Vmax™ Enriched Growth Media CL1500-1000 1000 mL $87
*Each reaction consists of one vial of Vmax Express Electrocompetent Cells and one 0.5 mL vial of Vmax Recovery Media. All products include one vial of a positive control plasmid.

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Vmax™ Express FAQs

Media and storage Questions

  • Can I culture Vmax™ Express on standard LB-agar?

    Plates containing either LB-Miller agar or brain heart infusion agar + v2 salts can be used. We typically use LB-Miller agar plates. Avoid LB-Lennox and LB-Luria, as these have less salt and do not support optimal growth.
  • Can I use richer growth media, such as Terrific Broth or Hyper Broth, to grow Vmax™ Express?

    Vmax™ Express grows well in a wide variety of carbon sources, growing better in media with higher levels of salt. Vmax™ cells have been successfully grown in TB (Terrific Broth), but we have not yet tested Vmax™ Express growth in Hyper Broth. Specific media recommendations can be found on the Vmax™ Express web page or in the Vmax™ Express User Guide.
  • Can I get good expression in ZYM/LB-IPTG?

    A number of media commonly used for E. coli are compatible with Vmax™ Express; however, Vmax™ Express grows best in higher osmotic conditions. Therefore, some media salt supplements may be required. A table with the suggested media is on the Vmax™ Express web page in the “How it Works” section and on page 8 of the Vmax™ Express User Guide. Recipes for suggested media are given on page 19 of the user guide. Pre-made Vmax™ Enriched Growth media is also available.
  • What selectable markers can I use with Vmax™?

    For antibiotic selection, we have demonstrated compatibility with antibiotics most commonly used for selection (i.e. Amp, Kan, Tet, Cm). A table with the specific antibiotics and the recommended concentrations is on the Vmax™ Express web page in the “How it Works” section and on page 9 of the Vmax™ Express User Guide.

General Questions

  • Is Vmax™ Express suitable for expressing large (~1000 aa) mammalian proteins?

    We have successfully expressed proteins up to 69 kDa in size with Vmax™ Express. A table showing examples of proteins expressed is available on the Vmax™ Express web page in the “Performance Data” section. Because amino acid compositions and protein properties are different between proteins, it is not possible to guarantee successful expression of a protein of any size. However, some proteins that are problematic to express in E. coli express well in Vmax™ Express.

Procedural Questions

  • Is heat shock transformation standard?

    Vmax™ Express is currently available only as an electrocompetent version. A chemicompetent version is in development.
  • We generally perform a cold shock immediately prior to induction. Could that be detrimental for Vmax™ Express?

    Avoid cold shock for Vmax™ Express and store agar plates with established Vmax™ Express cells at room temperature for up to 3 days. Single colonies will remain well isolated since Vmax™ Express cells exhibit contact inhibition. For long-term storage, we recommend preparing glycerol stocks with 25% glycerol and storing at −80°C. Do not store Vmax™ Express agar plates at 2–8°C as this can adversely impact re-proliferation.
  • Much of my work is performed in 96-well plates. Can I use 96-well plates to grow Vmax™ Express and express proteins?

    Although we routinely grow Vmax™ Express in 96-well deep well plates at room temperature without shaking, we have not yet tested protein expression in 96-well plates.

Questions about vectors

  • Which pET expression vector systems have been shown to work with Vmax™?

    A variety of common vectors and origins of replication are compatible with Vmax™. To date, T7 pET systems are the most studied expression vectors in Vmax™ Express. We have also had success expressing proteins in Vmax™ Express using pET28 and pET24. For more vector information on vectors and origins of replication compatible with Vmax™, see the Vmax™ Express web page or the Vmax™ Express User Guide.
  • Are Vmax™ Express cells compatible with standard E. coli expression vectors (e.g. pNIC) or will I need to clone into a new specialized expression vector?

    Vmax™ Express is compatible with vectors commonly used with E. coli and we have tested vectors with a variety of origins of replication. A table with this information is on the Vmax™ Express web page in the “How it Works” section and on page 10 of the Vmax™ Express User Guide. The pNIC plasmid has a pBR322 origin of replication, which we have shown to be compatible with Vmax™ Express.

Questions about Vmax Express in relation to E. coli

  • Is Vmax™ Express related to E. coli?

    Vmax™ Express and E. coli are gram negative prokaryotic bacteria. However, Vmax™ Express is derived from a different bacterial species and represents a novel host strain for molecular biology.
  • Do you have any data comparing protein expression in Vmax™ Express cells and E. coli?

    In nearly all instances we have examined to date, recombinant protein expression in Vmax™ Express cells is superior (e.g. expression is faster, proteins exhibit increased solubility, and larger quantities of are obtained in Vmax™ Express) in comparison to the expression of the same recombinant proteins in BL21 cells.
  • My protein is expressed as an inclusion body using E. coli. Will it be soluble in Vmax™ Express?

    We are able to express proteins in Vmax™ Express that do not express in E. coli. However, as each protein and set of expression conditions are unique, it is not possible to guarantee that a protein that is insoluble in E. coli will be soluble in Vmax™ Express. Regardless, Vmax™ Express is a novel host and may alleviate the solubility challenges associated with E. coli.
  • We find that protein expression in E. coli is improved with longer (overnight) and cooler (~20°C) expression incubations rather than maintaining cultures at 37°C after induction. Are there analogous data for Vmax™ Express?

    For optimal growth, express proteins in Vmax™ Express at 30°C. If needed, lower temperatures can be used as Vmax™ Express can grow at temperatures as low as room temperature and as high as 45°C.
  • How does Vmax™ codon usage compare to E. coli?

    Although Vmax™ has a different codon usage distribution than E. coli, the differences are minor. Proteins optimized for E. coli do not need to be re-optimized for Vmax™ Express. If you are preparing a new construct for expression in Vmax™ Express, codon optimization to Vmax™ Express may be beneficial. A codon usage table comparing Vmax™ with E. coli is available on the SGI-DNA website.
  • Should I modify my lysis and pelleting steps (shorter/longer sonication/centrifugation compared to BL21)?

    Like E. coli, Vmax™ Express is a gram-negative bacterium. Lysis conditions used for E. coli are sufficient. Vmax™ Express lysis does not require the addition of a nuclease since Vmax™ Express cells don’t exhibit viscosity due to the release of genomic DNA that tends to be troublesome in BL21(DE3)pLysS cells.
  • Why are proteins more soluble in Vmax™ Express?

    The specific properties of Vmax™ Express that allow proteins to be more soluble are not well understood at this time. Because Vmax™ is a different species, the intracellular environment is different from other bacterial strains used for protein expression. These differences appear to allow Vmax™ Express to produce high levels of soluble protein.

    Although we have observed that a number of proteins are highly expressed in active, soluble form in Vmax™ Express but are directed to insoluble inclusion bodies in E. coli, we do not expect that this will be the case for all proteins.