Pump Up Your Proteins
A novel chaperone-based strategy can increase yield of soluble recombinant protein
Editor's Note: This article first appeared in BIOforum Europe which is published by Git Verlag, a Wiley company.
The overproduction of heterologous proteins in bacteria frequently causes protein aggregation, resulting in the deposition of the desired protein in insoluble inclusion bodies. To increase the yield of soluble recombinant proteins, molecular chaperones-which assist protein folding and prevent protein aggregation in vivo-have been used often in biotechnology, albeit with limited success.
Recently, however, a novel two-step method involving the simultaneous overproduction of various cooperating chaperone systems, combined with an arrest of protein synthesis, increased the solubility of a large variety of client proteins. This method should be of general applicability in biotechnology.
The recombinant production of proteins of therapeutic and commercial value is frequently the only strategy that works to achieve sufficient amounts of the desired protein. The production is performed in host systems that include various cell lines, insect and yeast cells, or bacteria.
Bacterial systems like Escherichia coli can easily be genetically engineered and can produce large quantities of recombinant proteins in rapid, inexpensive fermentation processes. One major drawback of the E. coli system, however, is that the production of heterologous proteins is often accompanied by the failure of the recombinant protein to fold properly into its native structure. This causes deposition of the desired product into insoluble particles, called inclusion bodies (see Figure 1, p. 33).
The likelihood of protein aggregation is further increased by the use of strong promoters and high inducer concentrations, which lead to massive protein overproduction. Although inclusion body formation is beneficial for initial purification steps-because the protein of interest can be separated from most soluble contaminating proteins by centrifugation-regaining the functional native conformation of a protein is usually complicated and inefficient.
Protein misfolding and aggregation are not restricted to recombinant protein production; it's a challenge all cells face. Protein folding processes inside cells are complex. Nascent polypeptides emerging from the ribosome do not initially contain all the information necessary for folding and, therefore, transiently expose hydrophobic amino acids.
In the context of the crowded cellular environment, where macromolecule concentrations can reach 300-400 mg/ml, such exposure may cause inappropriate interactions that lead to protein misfolding and aggregation. Further, environmental stress-a sudden increase in temperature, for example-can cause proteins to unfold, increasing the likelihood of protein aggregation.
To optimize cellular protein folding and protect themselves from various stress conditions, all cells have developed protective systems. These systems consist of families of highly conserved proteins called molecular chaperones.1,2
Chaperones are found in high concentrations in the cells of all creatures, from bacteria to humans. Heat shock induces the synthesis of many chaperones, which are also called heat shock proteins (HSPs). Chaperones assist the de novo folding of proteins, prevent the aggregation of misfolded protein species, and repair damaged or aggregated proteins. The activity of chaperones relies on their ability to bind to short peptide segments enriched in hydrophobic amino acids. In native proteins, such peptide segments are found in the protein interior, the hydrophobic core. Nascent or misfolded polypeptides expose these segments and are therefore recognized by chaperones. This association protects non-native proteins from aggregation and supports their folding into the native state.
The cellular defense against protein misfolding and aggregation comprises several strategies. Holder chaperones, like the small heat shock proteins (sHSPs) prevent the formation of large protein aggregates. Folder chaperones, like the Hsp70 (DnaK) and the Hsp60 (GroEL) system, also interact with unfolded proteins, mediating their refolding in an ATP dependent process. Even aggregated proteins can be rescued by protein dis-aggregation and subsequent substrate refolding, mediated by a bi-chaperone system consisting of the Hsp70 (DnaK) system and the Hsp100 chaperone ClpB. The diverse chaperone classes form a functional network in which they cooperate to ensure maximum substrate re-folding. Figure 2 (below) summarizes the major cytosolic chaperones of E. coli and lists some of their crucial characteristics.
Chaperones assist the de novo folding of proteins, prevent the aggregation of misfolded protein species, and repair damaged or aggregated proteins. The activity of chaperones relies on their ability to bind to short peptide segments enriched in hydrophobic amino acids.
The application of molecular chaperones in biotechnology to increase the yield of properly folded recombinant client proteins has long been an attractive strategy. Introducing extra copies of chaperone encoding genes, together with the target gene, into the host cells ensures the simultaneous overexpression of the target and individual chaperone genes.
Some research has shown that co-expression of GroEL or DnaK increased the solubility of selected recombinant proteins to various extents.3 None of the individual chaperones tested, however, could be considered a general folding helper, one that could prevent mis-folding of any protein under question. Rather, it was shown that, for a variety of chaperones and different heterologous proteins used as substrates, specific chaperones work well for one substrate but do not work for others.
Two major factors are mainly responsible for limiting the application of chaperones in biotechnology. First, most studies only analyzed the effects of individual chaperones and didn't test for the simultaneous overproduction of various chaperone systems. Second, the continuous overproduction of recombinant proteins results in high levels of mis-folded protein species, favoring protein aggregation even in the presence of increased chaperone levels.
To bypass these restrictions, a recent study established a novel two-step chaperone-based procedure to increase the yield of soluble recombinant proteins.
The first step assesses the full potential of the cellular network of molecular chaperones for the production of soluble recombinant proteins(see Figure 3 above). Instead of co-expressing only individual chaperone systems, a set of compatible plasmids offers the possibility to co-produce the core of the E. coli quality control system, including the DnaK and GroEL chaperone systems, ClpB and IbpAB. These chaperone systems are well characterized and should build a cellular folding network that allows for the combined power of the major E. coli cytosolic chaperone systems in biotechnology.4
The second step was based on the initial observation that some inclusion bodies can be disintegrated by E. coli cells upon blocking the de novo synthesis of the aggregation-prone protein.5 By inhibiting the synthesis of the recombinant protein, either by removing the inductor molecule or by adding antibiotics that inhibit translation, it is possible to actively displace the equilibrium between protein aggregation and re-folding in vivo. Such treatment results in reduced levels of misfolded protein species and allows for more efficient chaperone-mediated protein refolding.
Protein folding processes inside cells are complex. Nascent polypeptides emerging from the ribosome do not initially contain all the information necessary for folding and, therefore, transiently expose hydrophobic amino acids.
To rigorously test the capacity of the novel approach, the two-step procedure was applied to 64 heterologous proteins that are difficult to produce in a soluble form using standard techniques. The novel strategy increased the solubility of 70% of the recombinant proteins tested up to 42-fold and was superior to standard chaperone applications, demonstrating the high efficiency and versatility of the two-step method.
The co-overproduction of the entire network of major cytosolic chaperones in E. coli cells, combined with a two-step procedure that allows for (re)folding of the heterologous proteins in the absence of ongoing de novo synthesis, resulted in the increased solubility of a large variety of recombinant proteins tested. The novel engineered E. coli strains, combined with the two-step strategy (patent application No. 10/500,883), should be applicable to a wide range of target proteins produced in biotechnology. �
1. Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science. 2002;295:1852-1858.
2. Bukau B, Weissman J, Horwich A. Molecular chaperones and protein quality control. Cell. 2006;125:443-451.
3. Schlieker C, Bukau B, Mogk A. Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology. J Biotechnol. 2002;96:13-21.
4. De Marco A, Deuerling E, Mogk A, et al. Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol. 2007;7:32.
5. Carrio MM, Villaverde A. Protein aggregation as bacterial inclusion bodies is reversible. FEBS Lett. 2001;489:29-33.