Cell-free production of aggregation-prone proteins in soluble and active forms

We have developed an efficient cell-free protein synthesis system for the production of soluble and active eukaryotic proteins that are predominantly produced as inclusion bodies in bacteria. S30 extracts (indicating the supernatant of cell homogenate when centrifuged at 30,000g) for cell-free protein synthesis were prepared from Escherichia coli that was modified to overexpress a set of chaperones (GroEL/ES or DnaK/J-GrpE) and disulfide isomerase (leader sequence-free mature DsbC expressed in the cytoplasm). The solubility and biological activity concentration (biological activity per unit volume of cell-free protein synthesis reaction mixture) of the protein synthesized by the new cell-free protein synthesis system showed a dramatic improvement. Solubility enhancement was most dramatic with the existence of DnaK/J-GrpE. It shows that the co-translational interaction with DnaK/J-GrpE prior to folding trial is important in maintenance of the aggregation-prone protein in a folding-competent soluble state. For maximizing the biological activity concentration of the expressed protein, the additional presence of GroEL/ES and DsbC was required. When human erythropoietin was expressed in the developed cell-free protein synthesis system including endogenously overexpressed chaperones and/or DsbC, the biological activity concentration of erythropoietin was enhanced by 700%. It implies that the post-translational folding and disulfide bond reshuffling as well as co-translational folding are important in acquiring functionally active protein from cell-free expression system. This is the first report of using S30 extracts including endogenously overexpressed chaperones and/or disulfide isomerase for the efficient production of soluble and active proteins in cell-free protein synthesis. This new cell-free protein synthesis system was capable of introducing much larger amounts of chaperones and disulfide isomerase compared to a conventional method that supplements them separately. The developed cell-free protein synthesis system supported efficient expression of the eukaryotic proteins in soluble and active forms without the need of any exogenous addition or coexpression of folding effectors.     

Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins in insect cells

The insect-baculovirus expression system has proved particularly useful for producing recombinant proteins that are biologically active. Overexpression of foreign proteins using the recombinant baculovirus system is often accompanied by aggregation of the overexpressed protein, which is thought to be due to a limitation of the translated protein folding in the infected cells. Co-infection of a recombinant baculovirus capable of expressing the human chaperone Hsp70 slightly increased the solubility of the overexpressed Epstein-Barr virus replication protein, BZLF1. Co-expression of Hsp70 and its co-factor, Hsdj or Hsp40, was here found to improve the solubility of the target protein several fold. Thus, a baculovirus expression system producing these molecular chaperones may find application for improved production of target foreign gene products in insect cells.     

A wheat germ cell-free system is a novel way to screen protein folding and function

For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two (15)N-labeled proteins and obtained (1)H-(15)N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and (1)H-(15)N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.     

Cell-free expression as an emerging technique for the large scale production of integral membrane protein

Membrane proteins are highly underrepresented in structural data banks due to tremendous difficulties that occur upon approaching their structural analysis. Inefficient sample preparation from conventional cellular expression systems is in many cases the first major bottleneck. Preparative scale cell-free expression has now become an emerging alternative tool for the high level production of integral membrane proteins. Many toxic effects attributed to the overproduction of recombinant proteins are eliminated by cell-free expression as viable host cells are no longer required. A unique characteristic is the open nature of cell-free systems that offers a variety of options to manipulate the reaction conditions in order to protect or to stabilize the synthesized recombinant proteins. Detergents or lipids can easily be supplemented and membrane proteins can therefore be synthesized directly into a defined hydrophobic environment of choice that permits solubility and allows the functional folding of the proteins. Alternatively, cell-free produced precipitates of membrane proteins can efficiently be solubilized in mild detergents after expression. Highly valuable for structural approaches is the fast and efficient cell-free production of uniformly or specifically labeled proteins. A considerable number of membrane proteins from diverse families like prokaryotic small multidrug transporters or eukaryotic G-protein coupled receptors have been produced in cell-free systems in high amounts and in functionally active forms. We will give an overview about the current state of the art of this new approach with special emphasis on technical aspects as well as on the functional and structural characterization of cell-free produced membrane proteins.     

Is Catalytic Activity of Chaperones a Selectable Trait for the Emergence of Heat Shock Response?

Although heat shock response is ubiquitous in bacterial cells, the underlying physical chemistry behind heat shock response remains poorly understood. To study the response of cell populations to heat shock we employ a physics-based ab initio model of living cells where protein biophysics (i.e., folding and protein-protein interactions in crowded cellular environments) and important aspects of proteins homeostasis are coupled with realistic population dynamics simulations. By postulating a genotype-phenotype relationship we define a cell division rate in terms of functional concentrations of proteins and protein complexes, whose Boltzmann stabilities of folding and strengths of their functional interactions are exactly evaluated from their sequence information. We compare and contrast evolutionary dynamics for two models of chaperon action. In the active model, foldase chaperones function as nonequilibrium machines to accelerate the rate of protein folding. In the passive model, holdase chaperones form reversible complexes with proteins in their misfolded conformations to maintain their solubility. We find that only cells expressing foldase chaperones are capable of genuine heat shock response to the increase in the amount of unfolded proteins at elevated temperatures. In response to heat shock, cells’ limited resources are redistributed differently for active and passive models. For the active model, foldase chaperones are overexpressed at the expense of downregulation of high abundance proteins, whereas for the passive model; cells react to heat shock by downregulating their high abundance proteins, as their low abundance proteins are upregulated.     

Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology

The amount of a native protein reflects an equilibrium of protein synthesis, de novo folding and protein stability. Stress situations, like heat shock, or overproduction of a protein can cause an imbalance in this equilibrium, resulting in protein aggregation. Molecular chaperones control protein folding processes and protect misfolded proteins from aggregation in all cells. Since protein aggregation is frequently observed upon synthesis of heterologous proteins in E. coli, molecular chaperones have been applied in biotechnology by their co-overproduction with the desired protein. While increasing protein solubility in some cases, this approach has not been generally successful. Recent findings demonstrate, that protein aggregation, even in case of inclusion bodies, must not be a dead end in the life cycle of a protein. Such resolubilization of aggregated proteins is mediated by a bi-chaperone system consisting of ClpB and DnaK, the prokaryotic representatives of the Hsp100 and Hsp70 families. The disaggregation capacity of this bi-chaperone system has now been demonstrated in vitro and in vivo for a wide variety of aggregated proteins and offers a new perspective to increase the solubility of proteins of interest.     

Co-translational involvement of the chaperonin GroEL in the folding of newly translated polypeptides

A large fraction of the newly translated polypeptides emerging from the ribosome require certain proteins, the so-called molecular chaperones, to assist in their folding. In Escherichia coli, three major chaperone systems are considered to contribute to the folding of newly synthesized cytosolic polypeptides. Trigger factor (TF), a ribosome-tethered chaperone, and DnaK are known to exhibit overlapping co-translational roles, whereas the cage-shaped GroEL, with the aid of the co-chaperonin, GroES, and ATP, is believed to be implicated in folding only after the polypeptides are released from the ribosome. However, the recent finding that GroEL-GroES overproduction permits the growth of E. coli cells lacking both TF and DnaK raised questions regarding the separate roles of these chaperones. Here, we report the puromycin-sensitive association of GroEL-GroES with translating ribosomes in vivo. Further experiments in vitro, using a reconstituted cell-free translation system, clearly demonstrate that GroEL associates with the translation complex and accomplishes proper folding by encapsulating the newly translated polypeptides in the central cavity formed by GroES. Therefore, we propose that GroEL is a versatile chaperone, which participates in the folding pathway co-translationally and also achieves correct folding post-translationally.     

Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems

Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.     

Between genotype and phenotype: protein chaperones and evolvability

Protein chaperones direct the folding of polypeptides into functional proteins, facilitate developmental signalling and, as heat-shock proteins (HSPs), can be indispensable for survival in unpredictable environments. Recent work shows that the main HSP chaperone families also buffer phenotypic variation. Chaperones can do this either directly through masking the phenotypic effects of mutant polypeptides by allowing their correct folding, or indirectly through buffering the expression of morphogenic variation in threshold traits by regulating signal transduction. Environmentally sensitive chaperone functions in protein folding and signal transduction have different potential consequences for the evolution of populations and lineages under selection in changing environments.     

促进原核表达蛋白可溶性的研究进展

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Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli

The enteric bacterium Escherichia coli is the most extensively used prokaryotic organism for production of proteins of therapeutic or commercial interest. However, it is common that heterologous over-expressed recombinant proteins fail to properly fold resulting in formation of insoluble aggregates known as inclusion bodies. Complex systems have been developed that employ simultaneous over-expression of chaperone proteins to aid proper folding and solubility during bacterial expression. Here we describe a simple method whereby a protein of interest, when fused in frame to the E. coli chaperones DnaK or GroEL, is readily expressed in large amounts in a soluble form. This system was tested using expression of the mouse prion protein PrP, which is normally insoluble in bacteria. We show that while in trans over-expression of the chaperone DnaK failed to alter partitioning of PrP from the insoluble inclusion body fraction to the soluble cytosol, expression of a DnaK–PrP fusion protein yielded large amounts of soluble protein. Similar results were achieved with a fragment of insoluble Varicella Zoster virus protein ORF21p. In theory this approach could be applied to any protein that partitions with inclusion bodies to render it soluble for production in E. coli.     

Productive interaction of chaperones with substrate protein domains allows correct folding of the downstream GFP domain

Green fluorescent protein (GFP) has been used to report protein folding by correlating solubility with fluorescence. In a GFP fusion protein, an upstream aggregation-prone domain can disrupt de novo folding of the GFP domain in Escherichia coli, resulting in a loss of fluorescence. Previously, we showed that prevention of misfolding of the upstream aggregation-prone domain by a coupled folding and binding interaction during protein synthesis restored both GFP fluorescence and solubility. Since molecular chaperones often fold nascent polypeptides through a bind-and-release interaction, the question remains whether the chaperone interaction with the upstream aggregation-prone domain enhances GFP fluorescence. Here, we demonstrate that a significant increase in GFP fluorescence occurred only when appropriate chaperones that recognized the aggregation-prone protein and helped its folding were co-expressed. A possible correlation between GFP fluorescence and the productive folding by chaperones is proposed. This study may provide a general strategy for identifying chaperones specific for difficult-to-fold proteins.     

Overexpression of human calnexin in yeast improves measles surface glycoprotein solubility

The limitations of high-level expression of virus surface proteins in yeast are not well understood. The inefficiency of yeast to produce active human virus surface glycoproteins, as well as other mammalian glycoproteins, is usually explained by the inefficient folding of the glycoprotein into its characteristic and functional three-dimensional structure from a random coil. The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. To improve folding and solubility of viral surface glycoprotein, the genes encoding human ER resident chaperones calnexin, calreticulin, immunoglobin binding protein (BiP), protein disulfide isomerase (PDI) and foldase (ERp57) were coexpressed together with hemagglutinin gene from measles virus in the yeast Saccharomyces cerevisiae. The effect of coexpressing chaperones on the total yield of measles virus hemagglutinin (MeH) as well as the intracellular fate of the glycoprotein was determined. Our results demonstrated that coexpression of human calnexin noticeably enhanced the quantity of the soluble glycosylated form of MeH in yeast. The coexpression of human calreticulin-, PDI-, ERp57- and BiP-encoding genes did not improve the quality of recombinant MeH.     

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.     

Removal of DnaK contamination during fusion protein purifications

The use of fusion proteins for recombinant protein expression in Escherichia coli has become popular because the carrier increases protein solubility, standardizes expression levels, and facilitates purification of the fusion products. However, we have observed that the peptide regions that fuse the carrier to the protein of interest bind E. coli Hsp70 molecular chaperones (DnaK) depending on their amino acid composition, resulting in an unwanted contamination during protein purification. Here we describe an approach that helps to circumvent this unwanted contamination. First, the appropriate amino acids surrounding and comprising the cloning site are chosen by using a software based on an algorithm already developed to decrease to a minimum the propensity of the fusion protein to bind DnaK. Second, DnaK contamination is significantly reduced by washing the fusion protein bound to the purification resin with MgATP plus soluble denatured E. coli proteins before elution. The approach can also be applied to eliminate other molecular chaperones.     

Improving the soluble expression of aequorin in Escherichia coli using the chaperone-based approach by co-expression with artemin

Abstract

Escherichia coli is a common host that is widely used for producing recombinant proteins. However, it is a simple approach for production of heterologous proteins; the major drawbacks in using this organism include incorrect protein folding and formation of disordered aggregated proteins as inclusion bodies. Co-expression of target proteins with certain molecular chaperones is a rational approach for this problem. Aequorin is a calcium-activated photoprotein that is often prone to form insoluble inclusion bodies when overexpressed in E. coli cells resulting in low active yields. Therefore, in the present research, our main aim is to increase the soluble yield of aequorin as a model protein and minimize its inclusion body content in the bacterial cells. We have applied the chaperone-assisted protein folding strategy for enhancing the yield of properly folded protein with the assistance of artemin as an efficient molecular chaperone. The results here indicated that the content of the soluble form of aequorin was increased when it was co-expressed with artemin. Moreover, in the co-expressing cells, the bioluminescence activity was higher than the control sample. We presume that this method might be a potential tool to promote the solubility of other aggregation-prone proteins in bacterial cells.     

A rapid protein folding assay for the bacterial periplasm

An array of genetic screens and selections has been developed for reporting protein folding and solubility in the cytoplasm of living cells. However, there are currently no analogous folding assays for the bacterial periplasm, despite the significance of this compartment for the expression of recombinant proteins, especially those requiring important posttranslational modifications (e.g., disulfide bond formation). Here, we describe an engineered genetic selection for monitoring protein folding in the periplasmic compartment of Escherichia coli cells. In this approach, target proteins are sandwiched between an N‐terminal signal recognition particle (SRP)‐dependent signal peptide and a C‐terminal selectable marker, TEM‐1 β‐lactamase. The resulting chimeras are localized to the periplasmic space via the cotranslational SRP pathway. Using a panel of native and heterologous proteins, we demonstrate that the folding efficiency of various target proteins correlates directly with in vivo β‐lactamase activity and thus resistance to ampicillin. We also show that this reporter is useful for the discovery of extrinsic periplasmic factors (e.g., chaperones) that affect protein folding and for obtaining folding‐enhanced proteins via directed evolution. Collectively, these data demonstrate that our periplasmic folding reporter is a powerful tool for screening and engineering protein folding in a manner that does not require any structural or functional information about the target protein.