Applications for protein expression

The Staby®Express application has been developed to produce a gene-of-interest in Escherichia coli. The application combines two technologies (T7 expression and Staby® plasmid stabilization) that allow high-yield protein expression and standardization of the production-protocol. We developed vectors with His-tag and/or GST-tag and Staby® adaptated CYS22 and SE1 competent bacteria.

Background

Plasmid instability is a significant concern in protein production. Typically, protein-production processes require the use of bacterial plasmids as vectors carrying the gene to be over-expressed. It has been demonstrated that the growth rate of plasmid-bearing cells is significantly reduced relative to that of a plasmid-free host, simply because protein production (corresponding to the gene-of-interest over-expression) represents a significant burden on cellular metabolism. Antibiotic-resistance genes are the most common selectable markers used in fermentation procedures to avoid plasmid-free cells to survive and dominate the culture. However, contamination of the product or biomass by antibiotics (or genes encoding an antibiotic resistance) is unacceptable from a medical or regulatory perspective (cf. FDA recommendations).

Our new stabilization system is based on the use of antidote/poison genes naturally found in plasmids, chromosomes, and bacteriophages. The ccdB selection gene codes for a small stable protein (about 100 amino acids) whereas the ccdA antidote gene codes for a small unstable protein (about 90 aa) that neutralizes the selection protein. These genes are extensively used in DNA cloning technology and are only active in enterobacteriacae (E. coli, Salmonella).

Principle

In our plasmid-stability system, the antidote gene is introduced in the plasmid DNA under the control of a constitutive promoter. On the other hand, the selection gene is introduced in the chromosome of the bacteria. Expression of this selection gene is under the control of a promoter strongly repressed by the antidote protein. Hence, when the plasmid is present in the bacteria, the poison is not produced. On the other hand, when the plasmid is lost, the antidote is degraded and the production of the toxin is induced, causing cell death.

The Staby®Express system is compatible with the use of an auto-inducible medium as StabyTMSwitch.

Higher plasmid stability = more proteins

Using Staby®Express, the plasmid is perfectly stable before and after the induction period. On the contrary, using the conventional strain BL21(DE3), the plasmid is not stably maintained . Consequently the production of the protein of interest is higher (3 to 5 times) with Staby®Express than using a conventional system with  antibiotics.  Note that no over-production of the antidote is detectable.

Services

We provide a fully integrated service from codon optimization to protein purification including this technology. We can adapt your vector or your strain of choice to use the StabyExpress in your production with a minimum of change.

Benefits of the Staby®Express application:

  • Higher yields of expressed proteins
  • Reduced background of non-target proteins
  • No need to transform the plasmid before each expression
  • Standardization of the protein-production protocol
  • No need to use antibiotics
  • System can be used in any culture medium

The Staby®Codon T7 application combines three technologies to ensure high-yield and standardized expression of eukaryote proteins in Escherichia coli. These technologies are (i) T7-controled expression, (ii) plasmid stabilization, and (iii)
codon-usage adaptation of E. coli for the efficient expression of proteins that contain rare codons. We developed vectors with His-tag and/or GST-tag and CYS22 and SE1 competent bacteria.

Principle

In all organisms, most amino acids are encoded by more than one codon: 61 codons are available for 20 amino acids. But each organism is characterized by a specific “codon bias” (see table), i.e. it preferentially uses some codons over others. In practice, when a heterologous gene is expressed in E. coli, this gene might exhibit some codons that are common in the original host but are rarely used in E. coli. Whereas, the presence of only a small number of rare codons might not severely depress target protein synthesis, the presence of clusters of and/or numerous rare codons generates a demand for one or more rare tRNAs. In turn, the rarity of some tRNAs leads to very low expression of the target protein due to premature translation termination, translation frameshifting, amino acid misincorporation, growth inhibition and plasmid instability. Six rare codons can cause problems in E. coli B (e.g.; BL21(DE3) or SE1): AGG and AGA (both encoding arginine using the argU tRNA), AUA (isoleucine, ileX tRNA), CUA (leucine, leuW tRNA), GGA (glycine, glyT tRNA), and CCC (proline, proL tRNA). An analysis of your gene-of-interest can be performed using Staby(TM)Soft.

In the StabyTMCodon T7 application, we solve the problem by the use of the pSCodon1 expression plasmid encoding the tRNA genes of the six rare codons. Hence, this plasmid both contains the T7 promoter for a strong expression and supplies the rare tRNAs. Moreover, this plasmid encodes the ccdA antidote gene. Consequently, transformed in bacteria containing the natural selection gene ccdB (such as the CYS22 and SE1 bacteria usedded in this application), the plasmid is stabilized. If some bacteria lose the vector, they will not obtain a selective (growth speed) advantage but will die (see the Staby®Express T7 application description). In practice, this additional stabilization technology solves the problem of plasmid instability and insures that during bacterial growth, 100% of the bacteria will carry the vector. Thus, the production of the protein of interest is higher and purer (lower amount of non-target proteins).

The Staby®Codon system is compatible with the use of an auto-inducible medium as StabyTMSwitch.

Results

To evaluate the efficiency of the Staby®Codon T7 application, a DNA fragment encoding a human protein containing 24 rare codons (10 prolines, 7 arginines, 3 isoleucines, 3 leucines and 1 glycine) was cloned into pStaby1 and pSCodon1. These plasmids were transformed in SE1. The protein is not or very poorly produced using pStaby1 but clearly visible using pSCodon1. The protein was purified and its integrity was confirmed by mass spectrometry.

Services

We provide a fully integrated service from codon optimization to protein purification including this technology. We can adapt your vector or your strain of choice to use the StabyExpress in your production with a minimum of change.

Benefits of the Staby®Codon application

  • High yield of heterologous-protein expression even when the protein contains rare codons;
  • Not necessary to mutate each rare codon;
  • Recombinant plasmid perfectly stabilized even without the use of antibiotics;
  • Reduced background of “parasite proteins;
  • No additional plasmid in the bacteria;
  • No “satellite” colonies obeserved even after extended incubation time

The CherryTMExpress application allows direct visualization (by eye!) of your protein of interest during protein production in E. coli and protein purification. No special requirements or reagents are needed. It is also
possible to quantify the protein concentration at any step by spectral measurement. The CherryTMExpress application combines multiple advantages: protein visualization, T7 expression and plasmid stabilization. See the movie on our homepage

Principle

The Cherry tag vectors allow direct visualization of your protein of interest during the whole process of protein production and purification without the need for any special requirements or reagents. The Cherry tag was incorporated into the StabyExpressTM T7 vector allowing high-level protein expression with or without antibiotics (click here for more info on the StabyTM technology). When using the Cherry tag vectors, your gene of interest is fused to a small sequence encoding a red polypeptide (heme binding part of cytochrome, 11 kDa) providing a visual aid for estimating expression level and solubility: bacteria expressing the fusion protein are red when the fused protein is soluble. The red color also constitutes a visual marker throughout protein purification. Concentration of the fusion protein can be determined easily by spectral measurement (before and after purification). In addition, the Cherry tag is highly soluble and thus, can increase the solubility of target proteins (for more information about protein solubility using CherryTM application, consult the application note). When using the Cherry tag as a N-terminal tag, it can be cleaved after purification using enterokinase (a recognition site is inserted at the C-terminal end of the tag sequence).
The CherryTMExpress system is compatible with the use of an auto-inducible medium as StabyTMSwitch.

Results

Using the Cherry tag vectors, recombinant protein expression is first detectable from the color of the bacterial pellet: the bacteria expressing a soluble fused protein are red. As lack of solubility is a major problem when expressing recombinant protein in E. coli, the Cherry tag system is convenient for rapid screening and optimization of protein solubility. The tag itself being highly soluble, it can increase the solubility of the target protein.

Furthermore, it is easy to visualize binding of the protein to the column (affinity or ion exchange) and to verify the absence of remaining protein of interest in the effluent. During elution, it is not necessary to collect multiple fractions and to analyse it to localize the target protein. Indeed, the Cherry tag system allows you to collect only the fraction containing the protein of interest.

When using the Cherry tag system, it is possible to quantify the protein concentration at any step (from protein production to the end of purification): a simple absorbance measurement at 413nm allows specific and accurate calculation of the target protein concentration.

Benefits of the CherryTM Tag system

  • Direct visualization of the protein of interest;
  • No need of any special apparatus or reagents;
  • Rapid screening and optimization of protein expression and solubility;
  • Easy quantification of the protein of interest;
  • High yield of protein production using StabyTM systems with or without antibiotics

The CherryTMCodon application allows direct visualization (by eye!) of your protein of interest during protein production in E. coli and protein purification. Special requirements or reagents are not needed. It is also possible to quantify the protein concentration at any step by spectral measurement. The CherryTMCodon application combines multiple advantages: protein
visualization, T7 expression, plasmid stabilization and codon-usage adaptation. See the movie on our home page

Principle

The Cherry tag vectors allow direct visualization of your protein of interest during the whole process of protein
production and purification without the need for any special requirements or reagents. When using the Cherry tag vectors, your gene of interest is fused to a small sequence encoding a red polypeptide (heme binding part of cytochrome, 11 kDa) providing a visual aid for estimating expression level and solubility: bacteria expressing the fusion protein are red when the fused protein is soluble. The red color also constitutes a visual marker throughout protein purification. Concentration of the fusion protein can be determined easily by spectral measurement (before and after purification). In addition, the Cherry tag is highly soluble and thus, can increase the solubility of target proteins (for more information about protein solubility using CherryTM kits, consult this application note). When using the Cherry tag as a N-terminal tag, it can be cleaved after purification using enterokinase (a recognition site is inserted at the C-terminal end of the tag sequence). The vector of the CherryTMCodon application (pSCherry2) encodes also rare t-RNAS allowing high-level expression of heterologous proteins (for more information, see the Staby®Codon application). Moreover, this vector is perfectly stabilized with or without antibiotics due to the action of the Staby® technology.

The CherryTMCodon system is compatible with the use of an auto-inducible medium as Staby®Switch.

Benefits:

  • Direct visualization of the protein of interest
  • No need of any special apparatus or reagents
  • Rapid screening and optimization of proteinexpression and solubility
  • Easy quantification of the protein of interest
  • Efficient expression of heterologuous genes due to Codon Correction
  • High yield of protein production using Staby® systems with or without antibiotics
  • Not necessary to mutate each rare codon;
  • Reduced background of “parasite proteins”
  • No additional plasmid in the bacteria

The Staby®Switch medium is an auto-inducible medium designed for high-level protein expression using Staby® products or any other IPTG-inducible bacterial expression system. Using Staby®Switch medium, protein expression is automatically induced when high cell density is reached. Thus, it is neither necessary to add IPTG nor to monitor optical density during bacterial growth. The medium is sterile and ready-to-use.

Principle

Staby®Switch is an auto-inducible medium: metabolization of medium components during bacterial growth will automatically induce protein expression at high cell density. Thus, it is neither necessary to add IPTG (isopropyl -β-D-thiogalactoside) nor to monitor optical density during bacterial growth. Furthermore, the target protein yield is often higher than using conventional IPTG induction. When using an autoinducible medium, it is essential to completely stabilize the plasmid encoding the protein of interest to obtain high expression yield. It is thus strongly recommended to use Staby® application (StabyExpress®, Staby®Codon, CherryTMExpress or CherryTMCodon) for high-yield protein expression. It is easier and faster to use Staby®Swicth than IPTG.

Results

The Staby®Switch medium is convenient for growing multiple cultures in parallel. Rapid growth to high densities is achieved (for maximizing the yield and efficiency of protein production) without the need to monitor bacterial growth nor to add inducer. As illustrated on the figure, expression yield is similar or higher using Staby®Switch than using conventional IPTG induction. Moreover, some proteins exhibit better conformation and solubility when using Staby®Switch instead of IPTG.

The Staby®Switch auto-inducible medium is a complete culture medium ready-to-use solution.

Benefits of the Staby®Switch medium

  • High yield of protein production using Staby® systems or other IPTG-inducible expression systems;
  • No IPTG required;
  • No need to monitor optical density (OD) during bacterial growth;
  • Faster expression protocol;
  • Overnight expression directly from a single colony or a glycerol stock;
  • Easy simultaneous expression of multiple clones for high-throughput applications