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Feb 1, 2011 (Vol. 31, No. 3)

Optimizing Expression in an E.coli System

Thermo Scientific MaxQ 8000 Shakers Allow All Flasks in an Experiment to Be Run Simultaneously

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    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.


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