Recombinant polypeptide production in E. coli: towards a rational approach to improve the yields of functional proteins
© de Marco; licensee BioMed Central Ltd. 2013
Received: 25 September 2013
Accepted: 30 October 2013
Published: 1 November 2013
The development of complementary technologies enabled the successful production of recombinant polypeptides in bacteria and opened to biology researchers new avenues as obtaining suitable amounts of proteins necessary for their experimental work became easy, fast, and inexpensive. Nevertheless, the recombinant approach remained somehow unpredictable, since many constructs resisted to apparent production or accumulated as aggregates.
Several factors and physical/chemical conditions that could improve the accumulation of native-like protein were identified. At the same time, it was acknowledged that the outcome of most of them was erratic and that almost any protein required its own specific optimized set of conditions to achieve its correct folding. The attempt to understand the critical points specific for recombinant protein production missed the goal of setting universally useful protocols, but contributed to the increase of the rate of success by proposing always new empiric combinations.
Nevertheless, the results published in the recent literature allow for a better comprehension of some key mechanisms controlling protein production in E. coli and could enable the elaboration of rational methodologies for improving the quantitative and qualitative features of the produced polypeptides. This result will be achieved when the identification of the limiting step that impairs the accomplishment of the native folding for any single construct will become straightforward. This minireview will discuss how factors such as the expression rate, the folding machinery, and the secretion efficiency may impact the final protein yields.
The intuition that recombinant expression could represent a metabolic burden for the host cell arose already in the infancy of this technology . Consequently, it was proposed to abandon the initial approaches that pushed the expression rates by exploiting high-copy number plasmids and strong (as well as leaky) promoters in favor of tighter control and slower expression rate obtained with suitable plasmids and reduced growth temperatures [2, 3]. For instance, in the lac promoter-regulated system both the addition of glucose and the overexpression of the lacI repressor and of the T7 lysozyme repress gene expression leakage effectively, impairing negative effects on plasmid segregation during cell replication . Recombinant expression can be regulated also by controlling the plasmid number per cell. This factor depends on the origin of replication sequences: whereas the pUC-based plasmids can accumulate in hundreds of copies per single cell, the pBR322-based plasmids produce tens of copies and the pSC101 and p15A systems just few.
Expressing less to fold better
The Walker strains were introduced as “mutant hosts that allow the synthesis of SOME membrane and globular proteins”  and for more than ten years the reasons of this (partial) success remained unknown. It was surprising to discover that the mutations present in the different strains were indeed convergent and involved a single mechanism, namely they contributed to the reduced efficiency of the lac UV5 promoter that controls the expression of T7 RNA polymerase [13, 14]. It was the first evidence that reducing the T7 RNA polymerase level in strains depending on this enzyme for recombinant expression could be a key element towards the control of the quality of the expressed protein and the increase of its accumulation. To assess such hypothesis, the control of the polymerase accumulation was obtained by means of the tunable expression of its inhibitor T7 lysozyme. The results confirmed that, in a specific range, there was an inverse correlation between polymerase availability and target protein accumulation . This output is in agreement with the very early supposition that reducing the expression rate could limit the cell metabolic burden and promote protein folding quality . The same effect of improved protein yield under conditions of promoter down-regulation was proved more recently for the arabinose-inducible expression system P(BAD) . A spontaneous and less efficient mutant allowed for lower cell toxicity and higher productivity of secreted membrane proteins.
From a practical point of view, the synthesis of fewer polypeptides per cell would result in more correctly folded proteins because the cell machinery would work more effectively, i.e. within its limits. This condition avoids the accumulation of unfolded polypeptides that tend to aggregate and to produce a toxic effect. Clearly, not all of the proteins have the same requirements. For instance, it has been estimated that only 85 of all the E. coli proteins have strict GroEL-dependence for completing their folding , whereas 180 strongly depend on DnaK and a minority need both chaperones . Therefore, the production of recombinant proteins that are either strictly GroEL-dependent or can use any/no chaperone to reach their native structure will exert a totally different pressure on the host cell in terms of competition for limiting components of the folding machinery. Both repressor leakiness and high expression rates can amplify the risk of accumulating misfolded proteins. The tight regulation of the recombinant expression is consequently crucial to limit the metabolic burden and the toxicity. From this perspective, it is interesting to learn that leakiness can be reduced 10-fold by a single mutation in the core domain of the lac repressor and that such mutation does not prevent IPTG binding and recombinant protein production . A complementary approach should investigate what chaperones are necessary for any particular protein to fold correctly in its original eukaryotic cell with the aim of overexpressing the prokaryotic homologues in the recombinant system. For instance, a systematic work has identified the substrates of human Hsp90  and such proteins could probably benefit from the co-expression of the E. coli orthologue chaperone ClpB when produced in bacteria.
Minimal metabolic perturbation
The production of transcripts originating from a single gene copy of the target DNA integrated in the chromosome represents a solution for obtaining sufficient yields of heterologous protein and minimizing the metabolic burden for the host cells. In such conditions, the cell physiology is not perturbed by the presence of high-copy number instable plasmids and by the massive production of antibiotic resistance enzymes . Sufficient protein yields are assured by enabling highly effective transcription and translation. Such result was obtained for the XylS/Pm expression system by combining several stimulatory elements in a unique expression cassette comprising the promoter, the 5′-untranslated mRNA region, and the XylS regulator coding sequence . The synergic effect of single point mutations in the sequences which regulate the transcription and translation steps allowed improving the final protein yields of 75 times. Both increased transcript production and its improved translation contributed to the final protein accumulation . Interestingly, the optimal level of the stimulation was protein-specific and higher transcriptional levels did not automatically correspond to higher yields of functional protein. These results were explained as the effect of the post-translational folding machinery saturation, a bottleneck that caused protein misfolding and aggregation. Such example underlines again how the final yields always depend on the limiting step in the long chain of events that leads from transcription to correctly folded proteins.
In the past it was also demonstrated that secreting thermodynamically favored constructs by the co-translational SRP route could facilitate the production of functional proteins because this option avoided the SecB-dependent accumulation in the cytoplasm of partially folded, inactive or toxic intermediates [26, 27]. Also the deletion of the gene responsible for the expression of the Trigger Factor (TF) chaperone contributed to limit the cell toxicity and to increase the accumulation of recombinant SRP-dependent proteins [16, 28]. Since TF binds 1:1 the polypeptides emerging from the ribosomes, it was argued that TF competes with the signal recognition particle for the nascent proteins. Therefore, in the absence of TF the cytoplasmic accumulation of non-productive protein intermediates would be drastically reduced. Nevertheless, too elevated expression rates would finally result in secretion impairment independently of the deletion strategy and of the chosen export route, since SecB and SRP share the same translocon unit  (Figure 2).
Summarizing, the improvement of the recombinant disulfide bond-dependent protein production relies on the capacity of calibrating the secretion and the translocon rates. The illustrated data emphasize also the limits of production strategies depending on the periplasmic accumulation of heterologous proteins. Even after successful export through the Sec-translocon, the proteins will undergo molecular crowding in the reduced periplasmic space and will be prone to aggregation .
Tuning protein aggregates
Aggregate formation is a constant of recombinant protein production. In contrast to previous hypotheses, we know now that aggregated proteins can conserve a consistent biological activity. Consequently, it is meaningful to study the aggregation process in vivo and to understand how chemical-physical factors can tune their characteristics  to improve their biotechnological value. Two opposite approaches have been proposed to cope with recombinant protein aggregation: try to limit it or to force the process and accumulate useful (functional and non-toxic) aggregates. The reversible dynamic of the aggregation-disaggregation process has been forced to improve the yields of soluble recombinant proteins by stimulating the bacterial cells to dissolve their inclusion bodies in vivo [7, 41]. As an alternative, aggregation has been exploited as a valuable means for accumulating functional recombinant protein in the inclusion bodies and simplifying its purification for biotechnological applications  (Figure 3). From this perspective, the identification of genetic backgrounds favorable for the production of particularly dense inclusion bodies is justified by the interest in improving the protein processing, obtaining higher titers in fermentation and simplified purification steps due to the reduced volumes and faster sedimentation. Pandey et al.  induced the production of recombinant model proteins in a bacterial library of randomly obtained knock-out mutants and succeeded in isolating clones producing more/denser inclusion bodies by means of percoll density gradient centrifugation. Unluckily, the authors did not evaluate whether the protein packed into denser inclusion bodies had structural and functional characteristics comparable to the control material or have lost them, as it has been observed when lipase A was fused to the self-assembling peptide ELK16 to improve its precipitation .
The biological value of proteins trapped in the inclusion bodies can be related to their physical structure rather than to their functional features. For instance, controlling the fermentation parameters allowed for the obtaining of inclusion bodies with variable geometries. When used to treat surfaces, they facilitate the adhesion of eukaryotic cells and promote tissue culture and differentiation .
Successful recombinant protein production is often strictly necessary to accumulate enough material for basic research studies as well as for biotechnological and clinical applications. A stimulating article  indicates that research stops where no reagents are available. Specifically, the authors remark that proteins that have a clear genetic link with human diseases, but for which reliable antibodies and chemical inhibitors are not accessible, are systematically less studied than similar proteins for which reagents are available. It would be interesting to analyze whether the reagent availability is related to the ease in obtaining functional and natively structured recombinant protein, since it can be expected that missing this step would represent a critical bottleneck. The knowledge accrued in the last 20 years conceives recombinant protein production as a long chain of successive events. Any of them can be limiting or can become the limiting after another obstacle has been removed. Therefore, the process optimization can be seen as a progressive removal of impairments: for a specific construct, too many transcripts blocks the translational machinery because there are not enough tRNA molecules for a specific codon, but in a strain overexpressing that tRNA the accumulated polypeptides can fail to fold due to DnaK shortage, and so on. Now we are probably approaching the comprehension of each single step involved in the whole process. For instance, several proteins that can improve protein secretion have been identified, the mechanisms by which low expression rate can lead to higher protein yields have been described in details, bacterial strains and vectors calibrated for working at physiological conditions are now available. The future efforts should be dedicated to a rational method to identify quickly and effectively the process weakness for any given protein to select the suitable counteraction. Combinatorial platforms exist for comparing experimentally protein production conditions by varying factors as different as bacterial strains, growth temperature, osmolyte concentration, fusion to different tags, promoter and transcription factor strength, mRNA stability and translation efficiency, chaperone co-expression, or signal peptide sequence [5, 7, 9, 47–52]. By contrast, predictive models are still missing. The lack of searchable data might be one of the reasons and, consequently, it would be beneficial for the community to make the effort of collecting and annotating experimental data in a standardized manner to facilitate their metadata analysis. Certainly, this effort requires the input of extra energy and can be perceived as a limitation of the research freedom, both reasons for the scarce success of previous initiatives . Nevertheless, such information is necessary to confirm some intuitions and to support statistically the cause-effect correlations evidenced by the already available results. The final accomplishment would be the development of holistic models able to consider all the elements that contribute to the protein production process.
This work was supported in part by the Creative Core AHA-MOMENT grant from Slovene Ministry of Science and European Fund for Regional Development and by Cross-Border Cooperation Programme Italy-Slovenia 2007–2013, (Project PROTEO, Code N. CB166). The author wishes to thank Alicja Gruszka for her comments.
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