Applications Manual

 

Reagents Needed but Not Provided: glycerol

 

The contents of the kit provide sufficient material to perform 40 media optimization tests or for use in establishing the other critical parameters that affect the expression of recombinant proteins in E. coli.

 

Protocols

 

Preparation of Media1. Dissolve the contents of each of the media packets in 1 liter of deionized water.
2. Add 4 ml of glycerol to the Turbo Broth™ and Power Broth™ solutions.
3. Dispense at desired volume into appropriate bottles or flasks.
4. Autoclave at 121°C for 15-20 min, depending on the volume per container, and allow to cool.
5. Dissolve the contents of the Glucose-Nutrient Mix in 100 mL deionized water.
6. Filter sterilize the Glucose-Nutrient Mix using a 0.2 mm filter.
7. Add 50 mL of the sterile Glucose-Nutrient Mix to a 1 liter of Hyper Broth™ and 20 mL to 1 liter of Glucose M9Y™ using aseptic technique.
8. Add sterile antibiotics as needed.

 

Media Screening Protocol1. Materials
1.1. 25 mL of each culture medium in 250 mL baffle bottomed flask
1.2. Wash Buffer: 50 mM sodium phosphate pH 7.5, 150 mM NaCl
1.3. 2x SDS-PAGE Loading Dye: 125 mM Tris-Cl pH 6.8, 4% SDS (w/v), 0.005% bromophenol blue (w/v), 20% glycerol (v,v), 5% b-mercaptoethanol (v,v)
1.4. Tris-Glycine SDS-polyacrylamide gel of appropriate composition
2. Methods
2.1. Inoculate a single colony of the recombinant strain into 10 mL LB Broth in a shake flask with baffle bottoms. Incubate at 37°C overnight.
2.2 Inoculate 25mL of each of the six media with 1mL of the overnight culture. Incubate the cultures at 37°C until the OD600 reaches 0.6.
2.3. Remove a 1mL sample ("pre-induction"), harvest the cells in a pre-weighed microfuge tube, and process as in step 2.7.
2.4. Add inducer (see Tip1) and continue incubating for 3 hours (see Tip 2).
2.5. Remove a 1 mL sample ("post-induction"), harvest the cells in a pre-weighed microfuge tube, and process as in step 2.7.
2.6 Harvest the remainder of the culture, wash with 10mL of wash buffer, determine the mass of the cell pellet, and store the cell pellets at -80°C. (see Tip 3)
2.7 Analyze for expression of the target protein as follows
2.7.1. To determine protein production per mL of culture:
2.7.1.1. Suspend the cell pellets from the pre- and post-induction samples in 500 mL water.
2.7.1.2. Mix 5 mL of each cell suspension with 15mL water and 5 mL 5x SDS-PAGE loading buffer. Heat at 100°C for 5 min. and load 10 mL per lane.
2.7.2. To determine the relative level of expression:
2.7.2.1. Suspend the cell pellets from the pre- and post-induction samples in water to a density of 10mg/mL.
2.7.2.2. Mix 5 mL of each cell suspension with 15mL water and 5mL 5x SDS-PAGE loading buffer. Heat at 100°C for 5 min and load 10mL per lane.
2.7.3. Stain the gel with Coomassie Blue, colloidal Coomassie Blue or silver stain. (see Tips 4 and 5)
3. Interpretation
3.1. After staining the gel, observe each lane and compare the “pre-induction” sample with the “post-induction” sample from each medium.  Elevated expression is indicated by the presence of a unique polypeptide band corresponding to the molecular mass of the target protein in the “post-induction” sample.
3.2. Compare the level of target protein obtained from cells grown in each of the six  media.  Select the medium which produces the highest level of target protein per ml of culture.  Figure 1 shows the results of a media screen experiment.
3.3 If two or more media give the same level of production per ml, then use the analysis of 2.7.2 to select the medium which gives the highest relative level of expression.

 

 

Figure 1. Media screen analysis by SDS-PAGE of a recombinant protein expressed in E. coli.  Arrow denotes the recombinant protein.  The protein was expressed in strain M15 grown in glucose M9Y, LB Broth, Power Broth™, Turbo Broth™, Superior Broth™ and Hyper Broth™, lanes 1 to 6, respectively

 

 

 

Tips of the TradeTip 1. The inducer used will depend on the expression system employed. The concentration of inducer is strain-dependent and the optimum concentration should be determined empirically.  For lacP-based expression systems, 1 mM IPTG is good for the media optimization.
Tip 2. Some recombinant proteins are expressed in E. coli as insoluble particles known as inclusion bodies.  The formation of inclusion bodies can not be predicted, but are indicated by the presence of intracellular refractive objects when viewed under oil immersion microscopy.3 The formation of inclusion bodies will not affect the results of the media screen, because the analysis is done on whole cell extracts prepared by boiling the cells in sodium dodecylsulfate.  This procedure completely denatures inclusion bodies as well as membrane and cytoplasmic proteins.  (In some instances lowering the temperature after induction can increase the amount of soluble protein recovered.  This should be determined experimentally.)
Tip 3. Once the medium yielding the highest level of expression has been determined, the cell paste can be used to prepare a small-scale extract.
Tip 4. Coomassie Blue stain should be sufficient to visualize the expression of a recombinant protein. Silver stain, while allowing detection of smaller amounts of protein, is more difficult to interpret and should only be used for examining whole cell extracts which are separated by SDS-polyacrylamide gels which are 20 cm in length or longer.  The long gel will give better resolution of individual polypeptide bands.
Tip 5. Alternative techniques can be applied to the media screen analysis.  Immunoblot or functional assays can be employed as appropriate.  Care should be taken when using functional assays by first demonstrating that some functional protein can be detected.  In most cases, the SDS-PAGE analysis is the method of choice during the early stages of developing the expression system.  Immunoblots should be used when the Coomassie blue stain does not reveal any expressed protein.

 

Technical Assistance
The scientific staff of the Athena Enzymes Systems™ group are specialists in the expression of recombinant proteins in microbial systems.  They have extensive expertise in all aspects of protein expression from the construction of expression vectors to the commercial production of recombinant proteins.  No matter what your question, please feel free to ask them for help.

 

 

 

Product Use Limitations
The Media Expression Kit™ was designed and is sold for research use only.  It should not be used for human diagnosis or drug use or administered to humans unless expressly cleared for that purpose by the appropriate regulatory authorities in the country of use.  All due care and attention should be exercised in the handling of the materials contained in the kit.

 

 

 

Product Warranty
AthenaES™ guarantees the quality and performance of the media contained in this kit for the cultivation of E. coli.  The suitability of a media formulation for a particular use is the responsibility of the end user.  No guarantee is made that a given protein will be expressed in any of the media formulations.  AthenaES will replace the product free of charge if it does not conform with the stated specifications.  Notice for replacement must be received within 60 days of opening the product.