Protein overexpression in Escherichia coli: Engineering bugs to make them more productive in protein synthesis and more robust when being cultured

---

By C. Perry Chou

Why Escherichia coli?
Proteins are useful bioproducts which frequently need to be produced in large amounts for scientific characterization or practical (such as industrial or medical) applications. Even with the development of the cell-free system, most campaigns in protein production will require a specific type of biological cell as the host.

Among various protein expression systems, including bacteria (Gram-positive or Gram-negative), fungi, insect cells, mammalian cells, plant cells and even the whole (transgenic) animals/plants, the Gram-negative bacterium of Escherichia coli is the most popular one. Two major factors result in its popularity.

First, the well-characterized genome and mature technologies (in genetic engineering, metabolic engineering, and protein engineering) for manipulation and construction of the host/vector system make it possible for a variety of attempts to improve expression performance. With a potential of reaching an expression level amounting to 50% of total cellular protein (10), E. coli is generally recognized as a protein overproducer.

Second, the fast growth rate and easy cultivation technology for E. coli make it suitable for many biomanufacturing applications. However, several drawbacks, such as the pathogenicity of this microorganism and lack of posttranslational processing abilities (particularly glycosylation) required for many eukaryotic proteins, prevent its full dominance as the expression host.

Nevertheless, its importance is still well received by industry. This is even true for therapeutic protein production, which typically has several options in selecting the expression system (1, 11), given the fact that nine out of the 31 therapeutic proteins approved during 2003 and 2006 are produced in E. coli, secondary to the mammalian cell systems (14).

Protein Targeting
Though cytoplasm is the compartment where all recombinant proteins are first synthesized and are possibly located as its final expression destination, some recombinant proteins are exported into the extracytoplasmic compartment, such as periplasm, inner or outer membrane or even extracellular medium, for various purposes (5). Periplasm has several advantages for expressing eukaryotic proteins because of its oxidative environment suitable for disulfide bond formation and the feasibility of obtaining proteins with authentic N-termini. This compartment also contains a less amount of total proteins that are isolated from the cytoplasm such that downstream purification will be facilitated. Microbial cell-surface display has been extensively explored due to its significant impact on various biotechnological and industrial applications, such as vaccine development, biosensor development, high-throughput screening of macromolecular libraries and preparation of whole-cell biocatalysts (9). Extracellular release of gene products offers an alternative for recombinant protein production in E. coli (6) and this approach is particularly valid for periplasmic proteins. The released proteins are less subject to intracellular proteolysis and selective secretion of the target protein would facilitate downstream purification. Also, reducing the local protein concentration via protein release can alleviate intracellular protein misfolding.

Gene Overexpression and Protein Recovery
Innovative design of genetically-engineered strains can highly increase the recombinant protein yield with minimum investment on the capital and operating costs. Figure 1 shows the major biological steps involved for recombinant protein synthesis in E. coli. Strategies for genetic construction of the expression vector are developed based on enhancing the efficiency of the major steps for heterologous gene expression, including replication, transcription and translation, though it is not uncommon that some of the posttranslational processing steps (particularly folding) could become limiting as well.

Various factors limiting gene expression have been reviewed extensively (10) and only a brief summary is given herein. For replication, the gene dosage can be increased with the use of high-copy-number or even 'run-away' plasmids and the plasmid instability needs to be properly maintained. Transcription is usually considered as the major step limiting the overall gene expression. As such, a number of strong promoter systems with various induction factors, such as chemicals (e.g. IPTG, arabinose or tetracycline), heat, pH and the availability of oxygen or carbon source, etc., have been developed to increase the transcriptional efficiency (perhaps translational efficiency as well) and in turn to increase the expression level. Translation can be limited by several factors, such as the initiation efficiency, the number of available ribosomes and/or tRNAs, the secondary structure and/or stability of mRNAs and the presence of rare codons, etc. Genetic modification of the regulatory elements to improve the translational efficiency has been proved effective.

Downstream processing is another major issue that should be taken into account when designing the expression vector. Several affinity tags, such as poly-His, GST, FLAG, cellulose binding domain, etc. have been developed to facilitate downstream purification of recombinant proteins (12). The expression vector is specifically designed such that the target protein would be expressed as a protein-tag fusion, which can be easily recovered using proper affinity chromatography. The target protein moiety can be released using a specific protease to cleave the junction between the target protein and the tag. Upon designing the expression vector, the above genetic strategies can be integrated to simultaneously enhance gene expression and protein purification (Figure 2).

Technical Limitations
Theoretically, all the biological steps depicted in Figure 1 have to be effective to result in a high recombinant protein yield. The step limiting the overall gene overexpression should be targeted for improving expression performance. However, enhancing the efficiency of the original limiting step could imply that another step becomes limiting. A typical example is that protein folding becomes limiting, resulting in the formation of misfolded protein, when strong promoters are used for boosting transcription. This raises an important issue that, for the overproduction of recombinant proteins, a 'balanced' protein synthesis flux throughout all the gene expression steps (i.e., transcription, translation and post-translational steps) should be properly maintained to avoid the accumulation of polypeptide intermediates in addition to boosting the individual limiting step.

Misfolding of the target gene products can result in the formation of insoluble protein aggregates as non-bioactive inclusion bodies in the cytoplasm or periplasm. This represents another major technical hurdle for recombinant protein production in E. coli (13). One of the reasons for inclusion body formation is that the overexpressed gene products cannot be suitably processed by folding modulators to develop the proper protein structure (2). In practice, these insoluble aggregates can be easily purified and refolding can be explored to regain the protein's biological activity (3). However, the potential of this approach is limited since renaturation of these misfolded proteins is often ineffective.

Note that the exploration of the above genetic strategies does not guarantee the improvement in the overall recombinant protein yield for the bioprocess due to another critical factor of cultivation. To optimize the culture performance, the two goals of high-cell-density cultivation and high-level gene expression are supposed to be achieved simultaneously. Theoretically, the maximum cell density that one can attain with fedbatch cultivation is approximately 200 g-dry-cell-weight (DCW)/L (8). On the other hand, accumulation of intracellular recombinant proteins at a level up to 50% of total cellular protein becomes achievable with the use of strong promoter systems (10). The presence of an excess amount of the foreign gene product in cells and the culture environment impact arising from high-level gene expression can present cells with significant physiological challenges.

High-level gene expression can make cells physiologically ill and stress-sensitive. The overexpressed foreign gene products can challenge cells with different levels of toxicity and metabolic burden, and cell growth is hindered as a result. On the other hand, due to the limitation in nutrient and/or oxygen availability, cells in dense cultures often have lower cellular activities and metabolic energy that are required for effective biosynthesis. A compromise in balancing the levels of gene expression and cell growth needs to be reached at a certain point to maximize the volumetric recombinant protein productivity. Studies have been conducted to characterize cellular responses to recombinant protein overproduction (7). Technical issues associated with physiological impact and engineering cell physiology for enhancing recombinant protein production have recently been reviewed (4).

Concluding Remarks
Most of the technical strategies developed for recombinant protein production in E. coli focus on innovative design of the expression vectors. However, the physiological deterioration associated with gene overexpression can result in a number of negative cellular responses, such as growth inhibition, cell lysis or even death, which are detrimental to protein productivity. To maximize the protein productivity, emphasis should also be given for high-cell-density cultivation by improving cell physiology upon gene overexpression. The integrative approaches in the aspects of both strain and bioprocess development for effective biomanufacturing form the major scope of C. Perry Chou's research activities.

References
1.     Andersen, D. C., and L. Krummen. 2002. Recombinant protein expression for therapeutic applications. Curr. Opin. Biotechnol. 13:117-123.
2.     Baneyx, F., and M. Mujacic. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22:1399-1408.
3.     Choe, W. S., R. Nian, and W. B. Lai. 2006. Recent advances in biomolecular process intensification. Chem. Eng. Sci. 61:886-906.
4.     Chou, C. P. 2007. Engineering Cell Physiology to Enhance Recombinant Protein Production in Escherichia coli. Appl. Microbiol. Biotechnol. in press.
5.     Cornelis, P. 2000. Expressing genes in different Escherichia coli compartments. Curr. Opin. Biotechnol. 11:450-454.
6.     Georgiou, G., and L. Segatori. 2005. Preparative expression of secreted proteins in bacteria: status report and future prospects. Curr. Opin. Biotechnol. 16:538-545.
7.     Gill, R. T., J. J. Valdes, and W. E. Bentley. 2000. A comparative study of global stress gene regulation in response to overexpression of recombinant proteins in Escherichia coli. Metab. Eng. 2:178-189.
8.     Lee, S. Y. 1996. High cell density culture of Escherichia coli. Trends Biotechnol. 14:98-105.
9.     Lee, S. Y., J. H. Choi, and Z. Xu. 2003. Microbial cell-surface display. Trends Biotechnol. 21:45-52.
10.     Makrides, S. C. 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60:512-538.
11.     Schmidt, F. R. 2004. Recombinant expression systems in the pharmaceutical industry. Appl. Microbiol. Biotechnol. 65:363-372.
12.     Terpe, K. 2003. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60:523-533.
13.     Villaverde, A., and M. M. Carrio. 2003. Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol. Lett. 25:1385-1395.
14.     Walsh, G. 2006. Biopharmaceutical benchmarks 2006. Nat. Biotechnol. 24:769-776.

Dr. C. Perry Chou is with the Department of Chemical Engineering, University of Waterloo, ON. He is an associate professor at UW and Canada Research Chair in Novel Strategy for High-Level Recombinant Protein Production. His primary research interest focuses on technological advancement in all the aspects for bioprocess development, including upstream (strain construction), midstream (cultivation technologies) and downstream processing (purification). In addition to academic research, he has developed several collaborative programs with private sectors for effective biomanufacturing.