Application Note 15
Top Ten Tips for Producing 13C/15N Protein in Abundance
Deborah A. Berthold, Victoria J. Jeisy, Terry L. Sasser, John J. Shea, Heather L. Frericks, Gautam Shah, and Chad M. Rienstra
Departments of Chemistry and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA
What could be easier than overexpressing an E. coli protein in E. coli? You don’t have to be an old hand at protein expression to know that this can often be more difficult than it sounds. We tested our skills recently with DsbA, a 20 kDa protein that catalyzes disulfide bond formation in the E. coli periplasm. The wildtype DsbA expressed well in LB medium and also in a BioExpress®-supplemented 13C/15N-labeling medium. Likewise, the DsbA C33S mutant expressed well in LB. But when we first tried to label C33S, our luck ran out – we saw no expression at all. Today we are producing 13C/15N DsbA C33S at a yield of 100 mg per liter. Here are our top ten tips for expressing recalcitrant proteins.
10. Stop the leaks. Leaky expression (i.e. expression in the absence of inducer) of a “toxic” protein or even a “less than healthful” mutant protein can slow cell growth, resulting in a suboptimal level of expression. In addition, because the half-life of ampicillin in a dense culture is less than 30 minutes, at later stages of growth there is a loss of selection for cells with ampicillin-resistant expression plasmids. So, any selection pressure stemming from leaky expression would give advantage to cells that have lost their expression plasmids. At best, the portion of plasmid-free cells in the culture at the time of induction are taking up 13C-labeled glucose without contributing to expression. At worst, leaky expression slows cell growth to the point that there is no growth at all in the labeling medium.
Promoters show variation in their leakiness, so use of a highly regulated promoter, such as the T7 promoter of the pET vector system, may be the solution. In our case, DsbA is on a high-copy plasmid (pUC119) and is expressed from a lac promoter (Kisigami, et al.,1995). In addition to this promoter being well-known for its leakiness, in optimizing the expression of the wildtype DsbA, we had changed E. coli strains. The original strain, E. coli M15/pREP4, contained additional copies of the lac repressor on pREP4; our preferred strain, E. coli C43(DE3), had no additional repressor other than the copy in the host genome. It was likely that the repressor binding sites on our expression plasmid titrated out all the copies of the repressor in the cell, leaving some promoters unrepressed. Our response: we put the compatible pREP4 plasmid into our E. coli C43(DE3) so that the cell would produce enough repressor for all the plasmid binding sites. Another solution might be to clone lacI q (a constitutive lac repressor) directly onto the expression plasmid; this is found, for example, in Qiagen’s QE80 series of His-tag expression vectors.
9. Slow down the train. For high levels of protein expression, the rate of transcription needs to be coupled to that of translation, which in turn needs to be coupled to any essential co- or post-translational events, such as folding, cofactor binding or membrane insertion. When transcription outstrips translation, loss of cell viability can occur (along with the destruction of ribosomal RNA and induction of proteases; see Dong, et al., 1995). There are several methods to tweak the rates of cellular metabolism to try to bring transcription, translation and post-translational processing in line. One can change promoters (Makrides, 1996; Baneyx, 1999). Strong promoters, such as T7, can be replaced with weaker promoters (arabinose, T5, tac). One can change cell lines. Two E. coli strains particularly suited for expression optimization, C41(DE3) and C43(DE3), were originally obtained from a selection for mutations that overcame lethality associated with overexpression from a T7 promoter (Miroux and Walker, 1996). These two strains also show an increase in plasmid stability relative to their parent BL21(DE3) strain (Dumon-Seignovert, et al., 2004). And, perhaps most easily, one can change the temperature during expression. For DsbA, the interplay between growth temperature and E. coli strain can be seen in Figure 1. Using BL21(DE3), slowing cell processes by growing at 25°C gives a large increase in expression over growth at 37°C (lanes 9-10 vs. lanes 7-8). In contrast, the strain C43(DE3), which is thought to have a mutation slowing the rate of transcription, gives a greater yield of expressed protein at 37°C (compare lanes 3-4 vs. 5-6). For DsbA C33S, we can choose between expressing at 37°C in C43 or expressing at 25°C in BL21(DE3). For some other proteins we have seen that both C43(DE3) and a lower temperature are required for the highest expression level.
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