February 1, 2011 (Vol. 31, No. 3)
Thermo Scientific MaxQ 8000 Shakers Allow All Flasks in an Experiment to Be Run Simultaneously
Recombinant proteins are an invaluable part of the life scientist’s toolbox and are increasingly being used as therapeutics. Arguably, the most commonly used expression host is Escherichia coli, a relatively simple and well-characterized system capable of producing large quantities of soluble protein in a short amount of time, without the need for extensive equipment or skills.
E. coli does have a couple of key drawbacks—it does not support post translational modifications (PTMs); and it is common for foreign genes to be poorly expressed, or for their protein products to become insoluble, forming inclusion bodies. This application note looks at the effect of varying temperature of induction (TOI), length of induction, and type of media used, using the Thermo Scientific MaxQ 8000 refrigerated stackable shaker from Thermo Fisher Scientific.
Low yields of soluble protein from E. coli have been tackled on multiple fronts. The use of expression and solubility tags has enabled the expression of many recombinant proteins; however, it is common for a recombinant protein to become insoluble once removed from its fusion tag.
Another focus of improving expression yield in E. coli has been codon usage, as there is a large difference in the codons most commonly used in eukaryotes compared to those preferred by E. coli. This has been tackled by two approaches, either through the use of E. coli strains that encode tRNAs that are rare in E. coli, but are used frequently in other organisms, or through the use of synthetic genes that have been codon optimized via complex algorithms that take into account the codon bias of E. coli.
Another distinct approach has been to empirically determine the best expression conditions for each individual recombinant. Variables that often have a large effect on the amount of soluble recombinant protein include TOI, length of induction, and type of media used. It is not directly clear how or why these factors are important. It is postulated that lower temperatures or general slower growth conditions increase the time that proteins have to fold, although this is not always born out.
Troubleshooting the best expression conditions for each individual recombinant protein often requires brute-force efforts to try multiple sets of variables. These solubility efforts can take weeks as variables are tried one by one.
This application note describes the expression screening experiment (Table) used to optimize the expression of a target protein using MaxQ® 8000 refrigerated stackable shakers to simultaneously test eight expression conditions. The target His-tagged recombinant protein is a 10.5 kDa, multistranded ß barrel with an intervening helix insert region. This protein is commercially significant to the Thermo Scientific Pierce product line and will be named protein X for this study. By using the MaxQ 8000 refrigerated stackable shakers we were able to test growth temperatures, length of induction, and effect of media in hours, rather than the many days normally needed to perform these studies.
The investigation was planned for maximum efficiency using a design of experiments process, to ensure that both the individual and combinatorial effects of each of the multiple experimental factors could be statistically analyzed.
Procedure
Protein X was expressed in E. coli from a plasmid under the control of a T7 promoter and the gene of interest.
A 100 mL overnight culture was grown at 37ºC. To scale up, 16 hours later a 10 mL aliquot of the overnight culture was added to each of eight, 2 liter baffled flasks. Four flasks contained 1 L of (Luria Bertani) LB media and four flasks contained 1 L of a super rich media that anecdotal data had shown improves expression of Protein X (50 mM Tris pH 7.5, 10 g yeast extract, 25 g tryptone, 5 g glucose, 5 g MgSO4).
All eight cultures were grown at 30ºC and were shaken at 200 rpm until O.D. 600 reached 0.5 (approximately three hours). At this point Isopropyl ß-D-1-thiogalactopyranoside (IPTG) was added to each flask to a final concentration of 200 µM. IPTG is a gratuitous inducer of the lac promoter, which is used to drive T7 polymerase expression in DE3 cells. The Protein X gene has a T7 promoter and is transcribed by T7 polymerase, allowing it to be overexpressed in the cell.
At this point, two LB and two super rich flasks were moved to a second shaker running at 15ºC and 200 rpm. The remaining four flasks were kept in the original shaker, at 200 rpm, but its temperature was lowered to 25ºC. (Flasks equilibrated to their new temperatures in less than 30 minutes, at this time-point protein expression of our gene of interest in undetectable).
At three hours, the cells from one flask at each condition (15ºC LB; 15ºC super rich, 25ºC LB; 25 ºC super rich) were harvested by centrifugation (four minutes @ 12,000 xg). The remaining four flasks were harvested the same way 16 hours after induction. Cell pellets were all frozen at -80ºC to aid lysis.
Cells were lysed in Thermo Scientific B-PER Bacterial Protein Extraction Reagent and enzymes and the lysate cleared by centrifugation (20 minutes @ 48,000 xg). Each lysate was then purified on a fresh 1 mL Thermo Scientific HisPur Cobalt Resin column using fast protein liquid chromatography at a flow rate of 2 mL/min. The column was washed with 20 mM Tris (pH 8.0 500 mM NaCl). Increasing concentrations of imidazole (10 mM, 20 mM, and 250 mM) were used to wash and then elute Protein X.
Fractions were analyzed by SDS gel electrophoresis (Figure 1) and quantitated by densitometry to determine the amount of protein X that had been purified. Measurements were performed in duplicate to assess error. The data was then analyzed statistically using the Design-Expert® 7 Workstation.
Figure 1. SDS PAGE gels that represent the findings of the study: The gels were all scanned in and the bands evaluated. NB—the insoluble fraction contains the majority of the expressed protein. Legend for lanes: 1—insoluble protein; 2, 3, 4, and 5—sequential elutions with 250 mM imidazole for sample incubated in super rich media at 25ºC with a 16 hour induction (flask 4); 6, 7, 8, and 9—sequential elutions with 250 mM imidazole for sample incubated in LB media at 25ºC with a 16 hour induction (flask 8).
Results
Using the Design-Expert 7 Workstation, all three factors (temperature, TOI, and media) were shown to have statistically important effects on the level of soluble protein X expression, as judged from the half-normal plot Figure 2. Half-normal plots compare the absolute values of ordered residuals from the data, to the expected values of ordered observations from a normal distribution to establish which experimental effects are important and which are unimportant.
Figure 2. Half-normal plot showing statistical relevance of the three experimental factors
Discussion
The procedure outlined in this article describes a method for the optimization of soluble protein expression in E. coli using the MaxQ 8000 refrigerated stackable shakers. Using the shakers we were able to grow cultures at different temperatures and study the effect of temperature, time, and media on the amount of soluble protein X produced. All three factors were seen to have statistically important effects on the level of soluble Protein X expression as judged from the half-normal plot. Interestingly, the effect of increasing both temperature and TOI was different for the two media. This experiment not only shows the importance of optimizing incubation parameters for recombinant protein expression in E. coli, but also that it can be achieved over hours rather than days when using good experimental design, coupled with superior equipment.
Key to the success of this investigation was the ability to design the experiment to run all flasks simultaneously using only two separate, stackable refrigerated shakers—the Thermo Scientific MaxQ 8000.
The MaxQ 8000 shaker range includes both refrigerated and incubated models and can be stacked (up to three units high), providing optimal use of any available floor space. In addition, the slide-out platform provides easy and rapid access to all the samples, during loading, unloading, and induction, for example. As a result, multiparameter, multilevel studies, such as the one demonstrated here, can be conducted very efficiently.
Figure 3. Plot of the effects of temperature, time of induction (TOI) using either Super Broth (A) or LB (B)
Mark Schofield ([email protected]) is research scientist II at Thermo Fisher Scientific.
